Compounds having selective activity for Retinoid X Receptors, and means for modulation of processes mediated by Retinoid X Receptors

ABSTRACT

Compounds, compositions, and methods for modulating processes mediated by Retinoid X Receptors using retinoid-like compounds which have activity selective for members of the subclass of Retinoid X Receptors (RXRs), in preference to members of the subclass of Retinoic Acid Receptors (RARs). Examples of such compounds are bicyclic benzyl, pyridinyl, thiophene, furanyl, and pyrrole derivatives. The disclosed methods employ compounds for modulating processes selectively mediated by Retinoid X Receptors.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/141,496, filed Oct.22, 1993, which is a continuation-in-part of the application Ser. No.08/052,051 filed on Apr. 21, 1993 now abandoned, which is acontinuation-in-part of the application Ser. No. 08/027,747 filed onMar. 5, 1993 now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/003,223 filed on Jan. 11, 1993 now abandoned,which is a continuation-in-part of application Ser. No. 07/944,783 filedon Sep. 11, 1992 now abandoned, which is a continuation-in-part ofapplication Ser. No. 07/872,707 filed Apr. 22, 1992 now abandoned, whoseentire disclosures are incorporated herein by reference. Benefit ofpriority under 35 U.S.C. §120 is claimed to these applications.

FIELD OF THE INVENTION

This invention relates to intracellular receptors and ligands therefor.More specifically, this invention relates to compounds having selectiveactivity for specific retinoic acid receptors, and methods for use ofsuch compounds.

BACKGROUND OF THE INVENTION

The vitamin A metabolite retinoic acid has long been recognized toinduce a broad spectrum of biological effects. A variety of structuralanalogues of retinoic a have been synthesized that also have been foundto be bioactive. Some, such as Retin-A® (registered trademark of Johnson& Johnson) and Accutane® (registered trademark of Hoffmann-LaRoche),have found utility as therapeutic agents for the treatment of variouspathological conditions. Metabolites of vitamin A and their syntheticanalogues are collectively herein called “retinoids”.

Synthetic retinoids have been found to mimic many of the pharmacologicalactions of retinoic acid. However, the broad spectrum of pharmacologicalactions of retinoic acid is not reproduced in full by all bioactivesynthetic retinoids.

Medical professionals have become very interested in the medicinalapplications of retinoids. Among their uses approved by the FDA is thetreatment of severe forms of acne and psoriasis. A large body ofevidence also exists that these compounds can be used to arrest and, toan extent, reverse the effects of skin damage arising from prolongedexposure to the sun. Other evidence exists that these compounds may beuseful in the treatments of a variety of severe cancers includingmelanoma, cervical cancer, some forms of leukemia, and basal andsquamous cell carcinomas. Retinoids have also shown an ability to beefficacious in treating premalignant cell lesions, such as oralleukoplakia, and to prevent the occurrence of malignancy.

Use of the retinoids is associated with a number of significant sideeffects. The most serious of these is that, as a class, they are amongthe most potent teratogens known. Teratogens are compounds that causesevere birth defects during specific periods of fetal exposure. Otherside effects include irritation of the tissues treated, which can be sosevere that patients cannot tolerate treatment.

Various investigations have been undertaken to elucidate thestructure-activity relationships governing the abilities of syntheticretinoids to induce the various pharmacological consequences of retinoicacid exposure. This has been a complicated task, however, since theassays available to investigators have been bioassays, carried outeither in intact animals or in isolated tissues. Technical constraintshave often dictated the use of different small animal species fordifferent assays. Interpretation of results has been complicated bypossible pharmacokinetic and metabolic effects and possible speciesdifferences in the receptors involved. Nevertheless, definitedifferences in the pharmacological effects of various syntheticretinoids have been observed.

Major insight into the molecular mechanism of retinoic acid signaltransduction was gained in 1988. Prior to that time, several highabundance cellular retinoid binding proteins were incorrectly inferredto be the signal transducing receptors for retinoic acid. In 1988, amember of the steroid/thyroid hormone intracellular receptor superfamily(Evans, Science, 240:889-95 (1988)) was shown to transduce a retinoicacid signal (Giguere et al., Nature, 330:624-29 (1987); Petkovich etal., Nature, 330: 444-50 (1987)). This unexpected finding relatedretinoic acid to other non-peptide hormones and elucidated the mechanismof retinoic acid effects in altering cell function. It is now known thatretinoids regulate the activity of two distinct intra-cellular receptorsubfamilies; the Retinoic Acid Receptors (RARs) and the Retinoid XReceptors (RXRs).

The first retinoic acid receptor identified, designated RAR-alpha, actsto modulate transcription of specific target genes in a manner which isligand-dependent, as has been shown to be the case for many of themembers of the steroid/thyroid hormone intracellular receptorsuperfamily. The endogenous low-molecular-weight ligand upon which thetranscription-modulating activity of RAR-alpha depends isall-trans-retinoic acid. Retinoic acid receptor-mediated changes in geneexpression result in characteristic alterations in cellular phenotype,with consequences in many tissues manifesting the biological response toretinoic acid. Two additional genes closely related to RAR-alpha wererecently identified and were designated RAR-beta and RAR-gamma and arevery highly related (Brand et al., Nature, 332:850-53 (1988); Ishikawaet al., Mol. Endocrin., 4:837-44 (1990)). In the region of the retinoidreceptors which can be shown to confer ligand binding, the primary aminoacid sequences diverge by less than 15% among the three RAR subtypes orisoforms. All-trans-retinoic acid is a natural ligand for the retinoicacid receptors (RARs) and is capable of binding to these receptors withhigh affinity, resulting in the regulation of gene expression. Thenewly-discovered retinoid metabolite, 9-cis-retinoic acid, is also anactivator of RARs.

A related but unexpected observation was made recently (Mangelsdorf etal., Nature, 345:224-29 (1990)), in which another member of thesteroid/thyroid receptor superfamily was also shown to be responsive toretinoic acid. This new retinoid receptor subtype has been designatedRetinoid X Receptor (RXR), because certain earlier data suggested that aderivative of all-trans-retinoic acid may be the endogenous ligand forRXR. Like the RARs, the RXRs are also known to have at least threesubtypes or isoforms, namely RXR-alpha, RXR-beta, and RXR-gamma, withcorresponding unique patterns of expression (Manglesdorf et al., Genes &Devel., 6:329-44 (1992)).

Although both the RARs and RXRs respond to all-trans-retinoic acid invivo, the receptors differ in several important aspects. First, the RARsand RXRs are significantly divergent in primary structure (e.g., theligand binding domains of RARα and RXRα have only 27% amino acididentity). These structural differences are reflected in the differentrelative degrees of responsiveness of RARs and RXRs to various vitamin Ametabolites and synthetic retinoids. In addition, distinctly differentpatterns of tissue distribution are seen for RARs and RXRs. For example,in contrast to the RARs, which are not expressed at high levels in thevisceral tissues, RXRα mRNA has been shown to be most abundant in theliver, kidney, lung, muscle and intestine. Finally, the RARs and RXRshave different target gene specificity. For example, response elementshave recently been identified in the cellular retinal binding proteintype II (CRBPII) and apolipoprotein AI genes which confer responsivenessto RXR, but not RAR. Furthermore, RAR has also been recently shown torepress RXR-mediated activation through the CRBPII RXR response element(Manglesdorf et al., Cell, 66:555-61 (1991)). These data indicate thattwo retinoic acid responsive pathways are not simply redundant, butinstead manifest a complex interplay. Recently, Heyman et al. (Cell,68:397-406 (1992)) and Levin et al. (Nature, 355:359-61 (1992))independently demonstrated that 9-cis-retinoic acid is a naturalendogenous ligand for the RXRs. 9-cis-retinoic acid was shown to bindand transactivate the RXRs, as well as the RARs, and therefore appearsto act as a “bifunctional” ligand.

In view of the related, but clearly distinct, nature of these receptors,ligands which are more selective for the Retinoid X Receptor subfamilywould be of great value for selectively controlling processes mediatedby one or more of the RXR isoforms, and would provide the capacity forindependent control of the physiologic processes mediated by the RXRs.Ligands which preferentially affect one or more but not all of thereceptor isoforms also offer the possibility of increased therapeuticefficacy when used for medicinal applications.

The entire disclosures of the publications and references referred toabove and hereafter in this specification are incorporated herein byreference.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, compositions, andmethods for modulating processes mediated by one or more Retinoid XReceptors. More particularly, the invention relates to compounds whichselectively or preferentially activate Retinoid X Receptors, incomparison to Retinoic Acid Receptors. These compounds selectivelymodulate processes mediated by Retinoid X Receptors. Accordingly, theinvention also relates to methods for modulating processes selectivelymediated by one or more Retinoid X Receptors, in comparison to RetinoicAcid Receptors, by use of the compounds of this invention. Examples ofcompounds used in and forming part of the invention include bicyclicbenzyl, pyridinyl, thiophene, furanyl, and pyrrole derivatives.Pharmaceutical compositions containing the compounds disclosed are alsowithin the scope of this invention. Also included are methods foridentifying or purifying Retinoid X Receptors by use of the compounds ofthis invention.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be better understood and its advantagesappreciated by those skilled in the art by referring to the accompanyingdrawings wherein:

FIG. 1 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 3-methyl-TTNCB.

FIG. 2 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by all-trans-retinoic acid.

FIG. 3 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 9-cis-retinoic acid.

FIG. 4 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 3-methyl-TTNEB.

FIG. 5 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 3-bromo-TTNEB.

FIG. 6 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 3-methyl-TTNCHBP.

FIG. 7 presents the standardized dose response profiles showing thetransactivation of RAR and RXR isoforms by 3-methyl-TTNEHBP.

FIG. 8 presents the inhibition of transglutaminase activity by9-cis-retinoic acid, all-trans-retinoic acid, and 3-methyl-TTNCB.

FIG. 9 presents the Topical Dose Response, based on the test on Rhinomice, for 9-cis-retinoic acid, all-trans-retinoic acid, 3-methyl-TTNCB,and 1,25-dihydroxy Vitamin D.

FIG. 10 presents the effect on rat HDL cholesterol of all-trans-retinoicacid, 9-cis-retinoic acid, 3-methyl-TTNCB, and 3-methyl-TTNEB.

FIG. 11 presents the concentration-related effect of 3-methyl-TTNEB andTTNPB individually on incorporation of radiolabeled thymidine into DNA.

FIG. 12 presents the concentration-related effect of a combination of3-methyl-TTNEB and TTNPB on incorporation of radiolabeled thymidine intoDNA.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses retinoid-like compounds or ligands which haveselective activity for members of the subfamily of Retinoid X Receptors(RXRs), in comparison to members of the subfamily of Retinoic AcidReceptors (RARs). Examples of such compounds are bicyclic benzyl,pyridinyl, thiophene, furanyl, and pyrrole derivatives which can berepresented by the formulae:

wherein:

R₁ and R₂, each independently, represent hydrogen or lower alkyl or acylhaving 1-4 carbon atoms;

Y represents C, O, S. N, CHOH, CO, SO, SO₂, or a pharmaceuticallyacceptable salt;

R₃ represents hydrogen or lower alkyl having 1-4 carbon atoms where Y isC or N;

R₄ represents hydrogen or lower alkyl having 1-4 carbon atoms where Y isC, but R₄ does not exist if Y is N, and neither R₃ or R₄ exist if Y isS, O, CHOH, CO, SO, or SO₂;

R′ and R″ represent hydrogen, lower alkyl or acyl having 1-4 carbonatoms, OH, alkoxy having 1-4 carbon atoms, thiol or thio ether, oramino, or R′ or R″ taken together form an oxo (keto), methano, thioketo,HO—N═, NC—N═, (R₇R₈)N—N═, R₁₇O—N═, R₁₇N═, epoxy, cyclopropyl, orcycloalkyl group and wherein the epoxy, cyclopropyl, and cycloalkylgroups can be substituted with lower alkyl having 1-4 carbons orhalogen;

R′″ and R″″ represent hydrogen, halogen, lower alkyl or acyl having 1-4carbon atoms, alkyl amino, or

R′″ and R″″ taken together form a cycloalkyl group having 3-10 carbons,and wherein the cycloalkyl group can be substituted with lower alkylhaving 1-4 carbons or halogen;

R₅ represents hydrogen, a lower alkyl having 1-4 carbons, halogen,nitro, OR₇, SR₇, NR₇, R₈, or (CF)_(n)CF₃, but R₅ cannot be hydrogen iftogether R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are all hydrogen, Z, Z′, Z″, Z′″, andZ″″ are all carbon, and R′ and R″ represent H, OH, C₁-C₄ alkoxy or C₁-C₄acyloxy or R′ and R″ taken together form an oxo, methano, orhydroxyimino group;

R₆, R₁₀, R₁₁, R₁₂, and R₁₃ each independently represent hydrogen, alower alkyl having 1-4 carbons, halogen, nitro, OR₇, SR₇, NR₇ R₈ or(CF)_(n)CF₃, and exist only if the Z, Z′, Z″, Z″″, or Z″″ from which itoriginates is C, or each independently represent hydrogen or a loweralkyl having 1-4 carbons if the Z, Z′, Z″, Z′″, or Z″″ from which itoriginates is N, and where one of R₆, R₁₀, R₁₁, R₁₂ or R₁₃ is X;

R₇ represents hydrogen or a lower alkyl having 1-6 carbons;

R₈ represents hydrogen or a lower alkyl having 1-6 carbons;

R₉ represents a lower alkyl having 1-4 carbons, phenyl, aromatic alkyl,or q-hydroxyphenyl, q-bromophenyl, q-chlorophenyl, q-fluorophenyl, orq-iodophenyl, where q=2-4;

R₁₄ represents hydrogen, a lower alkyl having 1-4 carbons, oxo, hydroxy,acyl having 1-4 carbons, halogen, thiol, or thioketone;

R₁₇ represents hydrogen, lower alkyl having 1-8 carbons, alkenyl(including halogen, acyl, OR₇ and SR₇ substituted alkenes), R₉, alkylcarboxylic acid (including halogen, acyl, OR₇ and SR₇ substitutedalkyls), alkenyl carboxylic acid (including halogen, acyl, OR₇ and SR₇substituted alkenes), alkyl amines (including halogen, acyl, OR₇ and SR₇substituted alkyls), and alkenyl amines (including halogen, acryl, OR₇and SR₇ substituted alkenes);

X is COOH, tetrazole, PO₃H, SO₃H, CHO, CH₂OH, CONH₂, COSH, COOR₉, COSR₉,CONHR₉, or COOW where W is a pharmaceutically acceptable salt, and whereX can originate from any C or N on the ring;

Z, Z′, Z″, Z′″and Z″″, each independently, represent C, S, O, N, but isnot Q or S if attached by a double bond to another such Z or if attachedto another such Z which is O or S, and is not N if attached by a singlebond to another such Z which is N;

n=0-3; and

the dashed lines in the second and seventh structures shown depictoptional double bonds;

or a pharmaceutically acceptable salt thereof.

As used in this disclosure, pharmaceutically acceptable salts includebut are not limited to: hydrochloric, hydrobromic, hydroiodic,hydrofluoric, sulfuric, citric, maleic, acetic, lactic, nicotinic,succinic, oxalic, phosphoric, malonic, salicylic, phenylacetic, stearic,pyridine, ammonium, piperazine, diethylamine, nicotinamide, formic,urea, sodium, potassium, calcium, magnesium, zinc, lithium, cinnamic,methylamino, methanesulfonic, picric, tartaric, triethylamino,dimethylamino, and tris(hydroxymethyl)aminomethane. Additionalpharmaceutically acceptable salts are known to those of skill in theart.

Representative derivatives according to the present invention includethe following:

-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-carbonyl)benzoic    acid, and designated “3-methyl-TTNCBN”;-   p(5,5,8,8-tetramethyl-,1,2,3,4-tetrahydro-3-isopropyl-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3-isopropyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid, and designated “3-XPR-TTNCB” or Compound 37;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-isopropyl-2-naphthyl-(2-methano)]-benzoic    acid, also known as    4-[1-(3-isopropyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic    acid, and designated “3-IPR-TTNEB” or Compound 42;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-ethyl-2-naphthyl-(2-methano)]-benzoic    acid, also known as    4-[1-(3-ethyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic    acid, and designated “3-ethyl-TTNEB” or Compound 45;-   p[(5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-bromo-2-naphthyl-(2-methano)]benzoic    acid, also known as    4-[1-(3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic    acid, and designated “3-bromo-TTNEB” or Compound 46;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3]-chloro-2-naphthyl-(2-methano)-benzoic    acid, also known as    4-[1-(3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl]ethenyl    benzoic acid, and designated “3-chloro-TTNEB” or Compound 43;-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-methano)]-benzoic    acid, also known as    4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl    benzoic acid, and designated “3-methyl-TTNEB”;-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-hydroxymethyl)]-benzoic    acid, also known as    4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)hydroxymethyl]benzoic    acid, and designated “3-methyl-TTNHMB”;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-bromo-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid, and designated “3-bromo-TTNCB” or Compound 41;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-chloro-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid, and designated “3-chloro-TTNCB” or Compound 38;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-hydroxy-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid, and designated “3-hydroxy-TTNCB” or Compound 39;-   p[5,5,8,8-tetramethyl-1,2,3,4-tetrahydro-3-ethyl-2-naphthyl-(2-carbonyl)]-benzoic    acid, also known as    4-[(3-ethyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid, and designated “3-ethyl-TTNCB” or Compound 40;-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-thioketo)]-benzoic    acid, also known as    4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)thioketo]-benzoic    acid, and designated “thioketone”;-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-carbonyl)]-N-(4-hydroxyphenyl)benzamide,    also known as    4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]-N-(4-hydroxyphenyl)benzamide,    and designated “3-methyl-TTNCHBP”;-   p[3,5,5,8,8-pentamethyl-1,2,3,4-tetrahydro-2-naphthyl-(2-methano)]-N-(4-hydroxyphenyl)benzamide,    also known as    4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]-N-(4-hydroxyphenyl)benzamide,    and designated “3-methyl-TTNEHBP” or Compound 63;-   2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-5-carboxylic    acid, designated “TPNEP” or Compound 58;-   ethyl    2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-5-carboxylate,    designated “TPNEPE” or Compound Et-58;-   2-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-5-carboxylic    acid, designated “TTNEP” or Compound 56;-   4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)epoxy]benzoic    acid, designated “TPNEB” or Compound 47;-   4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl]benzoic    acid, designated “TPNCB” or Compound 48;-   4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzene-tetrazole,    designated “3-methyl-TTNEBT” or Compound 55;-   5-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-2-carboxylic    acid, designated “TPNEPC” or Compound 60;-   2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl]pyridine-5-carboxylic    acid, designated “TPNCP” or Compound 62;-   methyl    2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl]-pyridine-5-carboxylate,    designated Compound Me-62;-   2-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl]pyridine-5-carboxylic    acid, designated Compound 111;-   4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid oxime, designated Compound 112; and-   4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic    acid methyloxime, designated Compound 115.

Representative structures for such compounds are as follows:

In addition, thiophene, furanyl, pyridine, pyrazine, pyrazole,pyridazine, thadiazole, and pyrrole groups function as isosteres forphenyl groups, and may be substituted for the phenyl group of the abovebicyclic benzyl derivatives.

Representative derivatives of the present invention can be preparedaccording to the following illustrative synthetic schemes:

Compounds of structure 1 containing R₅—lower alkyl are prepared inaccordance with U.S. Pat. No. 2,897,237. When R₅=Halo, Ohio, amino, orthio, the products are prepared by standard Friedel-Crafts reactionconditions combining the appropriate substituted benzene with2,5-dichloro-2,5-dimethyl hexane in the presence of aluminumtrichloride.

Condensation of 1 with mono-methyl terephthalate 2 was carried out byaddition of PCl₅ to 1 and 2 in CH₂Cl₂ followed by addition of AlCl₃ atroom temperature.

The resulting methyl esters 3 are hydrolyzed to the carboxylic acid 4 byrefluxing in aqueous KOH-MeOH followed by acidification.

Treatment of ketone 4 with NaBH₄ afforded alcohol 5.

Treatment of the methyl ester 3 with methyltriphosphonium bromide-sodiumamide in THF afforded the methano compound 6.

The carboxylic acid 7 was formed by adding KOH to methano compound 6MeOH, followed by acidification.

Treatment of the methyl ester 6 with hydrogen gas and 5% palladium overcarbon in ethyl acetate yields the hydrogenated compound 9.

Treatment of compound 9 with aqueous KOH in refluxing MeOH, followed byacidification, yields the carboxylic acid compound 10.

Condensation of 1 with thiophene 2,5-mono methyl dicarboxylic acid orfuranyl 2,5-mono methyl dicarboxylic acid was carried out by addition ofPCl₅ in CH₂Cl₂ followed by addition of AlCl₃ at room temperature to giveesters 11 and 12, which were hydrolyzed with KOH followed byacidification to the corresponding acids.

4,4-Dimethylchroman and 4,4-dimethyl-7-alkylchroman compounds of type 13and 14 as well as 4,4-dimethylthiochroman,4,4-dimethyl-7-alkylthiochroman,4,4-dimethyl-1,2,3,4-tetrahydroquinoline, and4,4-dimethyl-7-alkyl-1,2,3,4-tetrahydro-quinoline analogs weresynthesized by similar methods as compound 3, i.e., Friedel-Craftsconditions combining the appropriate dimethylchroman,dimethylthiochroman or dimethyltetrahydroquinoline with mono-methylterephthalate acid chloride in the presence of AlCl₃ or SnCl₄, followedby base hydrolysis and acidification to give the carboxylic acid. Forthe synthesis of the tetrahydroquinoline analogs, it was necessary toacylate the amine before Friedel-Crafts coupling with mono-methylterephthalate acid chloride. For the synthesis of the appropriatedimethylchromans, dimethylthiochromans and tetrahydroquinolines, seeU.S. Pat. Nos. 5,053,523 and 5,023,341 and European Patent PublicationNo. 0284288.

Compounds of the type 18 were synthesized by nucleophillic addition ofthe Grignard reagent 16 to bromotetralone, bromoindane, or otherbicyclic ketone derivative. Treatment of the resulting alcohol withmethanolic HCl gave the intermediate 17. Displacement of the brominewith CuCN in quinoline gave the nitrile which was then hydrolyzed to theacid 18 in refluxing KOH. Bromine compound 15 was synthesized from2,5-dichloro-2,5-dimethylhexane and 2-bromotoluene with a catalyticamount of AlCl₃. Treatment of compounds 3-methyl-TTNCB and3-methyl-TTNEB with DCC, p-aminophenol, and DMAP resulted in theamino-esters 19 and 20.

Representative pyridinal derivatives (compounds 21, 23, 26, and 27) maybe prepared according to the illustrative synthetic schemes shown above.The synthesis of compound 21 is similar to that previously described forcompound 7. Pentamethyl tetrahydronaphthalene 1, pyridinal acid chloride24, and AlCl₃ are stirred in CH₂Cl₂ to give the ketone 25. Treatment ofthe ketone 25 with methyl triphosphonium bromide-sodium amide in THFafforded the ethenyl compound 26. Hydrolysis of 26 (KOH, MeOH) followedby acidification gave the acid 21. The cyclopropyl analog 23 wassynthesized by treatment of the ethenyl compound 26 with CH₂I₂, zincdust, CuCl in refluxing ether (Simmons-Smith reaction). Hydrolysis ofthe resulting cyclopropyl ester 27 was achieved with methanolic KOHfollowed by acidification to give compound 23. When R₁-R₅ are methyl,for example, compound 62 (TPNCP) is obtained, as shown in Example 33below.

Other cyclopropyl derivatives such as TPNCB (compound 48) may belikewise prepared by the same method as described for analog 23: olefin6 is treated with the Simmons-Smith reagent described above, followed byhydrolysis with methanolic-KOH and acidification (HCl) to give thedesired cyclopropyl derivative. Epoxy derivatives such as TPNEB(compound 47) may be synthesized by treatment of compound 7 withm-chloroperbenzoic acid at room temperature in CH₂Cl₂ for several hours.

Alternatively, pyridinal analogs, such as compounds 58 (TPNEP), 60(TPNEPC), and 61 (3TTNEPE), may be prepared by the following syntheticroute.

Synthesis of Oximes and Methyloximes

Oxime from Carboxylic Acid:

Oximes from Esters:

Methyloximes:

Synthesis of Alkyloximes

Synthesis of Cyanoimine

Representative oxime derivatives (compounds 112, 113, and 114) may beprepared according to the illustrative synthetic schemes shown above.Synthesis of a representative methyloxime (compound 115) is also shown.A ketone such as 3-methyl-TTNCB is treated with hydroxylaminehydrochloride in pyridine and heated at reflux to give oxime 112. Alkyloxime ethers are prepared from the corresponding ketone (such as3-methyl-TTNCB) by treatment with methoxylamine hydrochloride inrefluxing pyridine to give the methoxyomine 116. Also see Examples44-49, below. Et-115 (and other ethyl esters such as Et-58, Et-62,Et-3-methyl-TTNEB) can be made by treatment of the respective carboxylicacids with oxalyl chloride to form the acid chloride, followed bytreatment with EtOH and pyridine to give the ethyl ester. The oximeEt-112 can be made fromEt-4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoate(Et-3-methyl TTNCB) by treatment with pyridine/EtOH and NH₂OH—CHl atreflux.

Substituted oximes (compounds 138-143) may also be prepared as shownabove. These compounds were synthesized from the corresponding freeoxime (112) by treatment of the oxime with NaH followed by alkylationwith the appropriate bromo alkyl group (R—Br).

Illustrative examples for the preparation of some of the compoundsaccording to this invention are as follows:

EXAMPLE 1 Preparation of compound 3 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′ and R″ are oxo, and X=COOMe

To 7 gm (34.7 mmol) of1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene and 6 gm (33.3 mmol)of mono-methyl teraphthalate in 200 mL of CH₂Cl₂ was added 8 g (38.8mmol) of PCl₅. The reaction boiled vigorously and turned clear within 10min. After stirring for an additional 1 h, 6 g (43.5 mmol) of AlCl₃ wasadded in 1 g portions over 15 min. and the reaction was allowed to stirovernight. The mixture was poured into 300 mL of 20% aqueous HCl andextracted with 5% EtoAc-hexanes, dried (MgSO₄), concentrated, andcrystallized from MeOH to give ca. 6 gm (16.5 mmol) of methyl ester 3.¹HNMR (CD₃ OCD₃) δ 1.20 (s, 2(CH₃)), 1.35 (s, 2(CH₃)), 1.75 (s, 2(CH₂)),2.31 (s, CH₃), 3.93 (s, COOCH₃), 7.21 (s, Ar—CH), 7.23 (s, ArCH), 7.85(d, J=8 Hz, Ar-2(CH)), 8.18 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 2 Preparation of compound 4 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′ and R″ are oxo, and X=COOH (3-methyl-TTNCB)

To 6 gm (16.5 mmol) of methyl ester 3 suspended in 100 mL of MeOH wasadded 50 mL of 5N aqueous KOH. The mixture was heated under reflux for 1h, cooled, acidified (20% aqueous HCl) and the organics extracted withEtOAc. After drying (MgSO₄), the product was concentrated andprecipitated from 1:4 EtOAc-hexanes to give ca. 5 g (14.3 mmol) of acid4. ¹HNMR (CD₃OCD₃) δ 1.20 (s, 2(CH₃)), 1.35 (s, 2(CH₃)), 1.75 (s,2(CH₂)), 2.31 (s, CH₃), 7.21 (s, Ar—CH), 7.23 (s, Ar—CH), 7.91 (d, J=8Hz, Ar-2(CH)), 8.21 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 3 Preparation of compound 5 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′=H and R″=OH, and X=COOH (3-methyl-TTNHMB)

To a 1:1 THF-MeOH solution containing 1 g (2.86 mmol) of ketone 4 wasadded 100 mg of NaBH₄. The mixture was heated to 50° C. for 10 min.,cooled, acidified (20% aqueous HCl), and the organics extracted (EtOAc).After drying (MgSO₄), the product was concentrated and precipitated from1:3 EtOAc-hexanes to give 550 mg (1.56 mmol) of the alcohol 5. ¹HNMR(CD₃OCD₃) δ 1.20 (s, CH₃)), 1.22 (s,(CH₃)), 1.22 (s, 2(CH₃)), 1.65 (s,2(CH₂)), 2.21 (s, CH₃), 6.00 (s, —CHOH—), 7.09 (s, Ar—CH), 7.41 (s,Ar—CH), 7.53 (d, J=8 Hz, Ar-2(CH)), 8.01 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 4 Preparation of compound 6 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′ and R″ are methano, and X=COOMe

To 1 gm of methyl ester 3 (2.7 mmol) in 25 mL of dry THF was added 1.2 g(3.08 mmol) of methyltriphosphonium bromide-sodium amide. The solutionwas stirred at RT for 3 h or until complete by TLC (20% EtOAc-hexanes).Water was added and the organics were extracted with EtOAc, dried(MgSO₄), concentrated and purified by SiO₂ chromatography (5%EtOAc-hexanes) followed by crystallization from MeOH to give 700 mg(1.93 mmol) of methano compound 6. ¹HNMR (CD₃OCD₃) δ 1.22 (s, 2(CH₃)),1.30 (s, 2(CH₃)), 1.72 (s, 2(CH₂)), 1.95 (s, CH₃), 3.85 (s, COOCH₃),5.29 (s, ═CH), 5.92 (s, ═CH), 7.19 (s, Ar—CH), 7.20 (s, Ar—CH), 7.39 (d,J=8 Hz, Ar-2(CH)), 7.96 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 5 Preparation of compound 7 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′ and R″ are methano, and X=COOH (3-methyl-TTNEB)

To 500 mg of methano compound 6 (1.38 mmol) in 20 mL of MeOH was added 5mL of 5 N aqueous KOH and the suspension was refluxed for 1 h. Afteracidification (20% aqueous HCl) the organics were extracted (EtOAc),dried (MgSO₄), concentrated, and the solids recrystallized fromEtOAc-hexanes 1:5 to give 350 mg (1.0 mmol) of the carboxylic acid 7.¹HNMR (CD₃OCD₃), δ 1.22 (s, 2(CH₃)), 1.30 (s, 2(CH₃)), 1.72 (s, 2(CH₂)),1.95 (s, CH₃), 5.22 (s, ═CH), 5.89 (s, ═CH), 7.19 (s, Ar—CH), 7.20 (s,Ar—CH), 7.39 (d, J=8 Hz, Ar-2(CH)), 7.96 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 6 Preparation of compound 37 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is isopropyl, R′ and R″ are oxo, and X=COOH (3-IPR-TTNCB)

The compound was prepared in a manner similar to that of compound 4except that6-isopropyl-1,1,4,4-tetramethyl-1,2,3,4-tetra-hydronaphthalene wassubstituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene inexamples 1 and 2. MP: 254° C.; ¹H-NMR (CDCl₃) δ 1.19 (d, J=7 Hz,CH(CH₃)₂), 1.21 (s, 2(CH₃)), 1.33 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 3.12(q, J=7 Hz, CH(CH₃)₂), 7.14 (s, Ar—CH), 7.37 (s, Ar—CH), 7.92 (d, J=8Hz, Ar-2(CH)), 8.18 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 7 Preparation of compound 38 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is chloro, R′ and R″ are oxo, and X=COOH (3-chloro-TTNCB)

The compound was prepared in a manner similar to that of compound 4except that 6-chloro-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1 and 2. MP: 254° C.; ¹H-NMR (CDCl₃) δ 1.26 (s, 2(CH₃)),1.32 (s, 2(CH₃)), 1.72 (s, 2(CH₂)), 7.35 (s, Ar—CH), 7.36 (s, Ar—CH),7.91 (d, J=8 Hz, Ar-2(CH)), 8.19 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 8 Preparation of compound 39 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is hydroxy, R′ and R″ are oxo, and X=COOH (3-hydroxy-TTNCB)

The compound was prepared in a manner similar to that of compound 4except that 6-hydroxy-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1 and 2. MP: 264° C.; ¹H-NMR (CDCl₃) δ 1.17 (s, 2(CH₃)),1.31 (s, 2(CH₃)), 1.68 (s, 2(CH₂)), 7.02 (s, Ar—CH), 7.44 (s, Ar—CH),7.77 (d, J=8 Hz, Ar-2(CH)), 8.27 (d, J=8 Hz, Ar-2(CH)), 11.50 (s, —OH).

EXAMPLE 9 Preparation of compound 40 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is ethyl, R′ and R″ are oxo, and X=COOH (3-Et-TTNCB)

The compound was prepared in a manner similar to that of compound 4except that 6-ethyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1 and 2. MP: 226° C.; ¹H-NMR (CDCl₃) δ 1.16 (t, J=7.5 Hz,—CH₂CH₃), 1.19 (s, 2(CH₃)), 1.32 (s, 2(CH₃)), 1.69 (s, 2(CH₂)), 2.69(q,J=7.5 Hz, CH₂CH₃), 7.20 (s, Ar—CH), 7.25 (s, Ar—CH), 7.87 (brd,Ar-2(CH)), 8.20 (brd, Ar-2(CH)).

EXAMPLE 10 Preparation of compound 41 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is bromo, R′ and R″ are oxo, and X=COOH (3-bromo-TTNCB)

The compound was prepared in a manner similar to that of compound 4except that 6-bromo-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1 and 2. MP: 275° C.; ¹H-NMR (CDCl₃) δ 1.25 (s, 2(CH₃), 1.32(s, 2(CH₃)), 1.71 (s, 2(CH₂)), 7.30 (s, Ar—CH), 7.54 (s, Ar—CH), 7.90(d, J=8 Hz, Ar-2(CH)), 8.18 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 11 Preparation of compound 42 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is isopropyl, R′ and R″ are methano, and X=COOH (3-IPR-TTNEB)

The compound was prepared in a manner similar to that of compound 7except that6-isopropyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalene wassubstituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene inexamples 1, 2, 4, and 5. MP: 252° C.; ¹H-NMR (CDCl₃) δ 1.05 (d, J=7 Hz,CH(CH₃)₂), 1.27 (s, 2(CH₃)), 1.32 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 2.73(q,J=7 Hz, CH(CH₃)₂), 5.32 (s, ═CH), 5.87 (s, ═CH) 7.06 (s, Ar—CH), 7.23(s, Ar—CH), 7.40 (d, J=8 Hz, Ar-2(CH)), 8.040 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 12 Preparation of compound 43 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is chloro, R′ and R″ are methano, and X=COOH (3-chloro-TTNEB)

The compound was prepared in a manner similar to that of compound 7except that 6-chloro-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1, 2, 4, and 5. MP: 233° C.; ¹H-NMR (CDCl₃) δ 1.28 (s,2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s, 2(CH₂)), 5.42 (s, ═CH), 5.89 (s,═CH), 7.23 (s, Ar—CH), 7.28 (s, Ar—CH), 7.37 (d, J=8 Hz, Ar-2(CH)), 8.03(d, J=8 Hz, Ar-2(CH)).

EXAMPLE 13 Preparation of compound 44 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is hydroxy, R′ and R″ are methano, and X=COOH(3-hydroxy-TTNEB)

The compound was prepared in a manner similar to that of compound 7except that 6-hydroxy-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1, 2, 4, and 5. MP: 216° C.; ¹H-NMR (CDCl₃) δ 1.21 (s,2(CH₃), 1.30 (s, 2(CH₃)), 1.68 (s, 2(CH₂)), 5.54 (s, ═CH), 5.94 (s,═CH), 6.86 (s, Ar—CH), 7.00 (s, Ar—CH), 7.48 (d, J=8.4 Hz, Ar-2(CH)),8.07 (d, J=8.4 Hz, Ar-2(CH)).

EXAMPLE 14 Preparation of compound 45 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is ethyl, R′ and R″ are methano, and X=COOH (3-Et-TTNEB)

The compound was prepared in a manner similar to that of compound 7except that 6-ethyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1, 2, 4, and 5. MP: 236° C.; ¹H-NMR (CDCl₃) δ 0.99 (t, J=7.6Hz, —CH₂CH₃), 1.27 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.70 (s, 2(CH₂)),2.29(q, J=7.6 Hz, —CH₂CH₃), 5.34 (s, ═CH), 5.83 (s, ═CH), 7.08 (s,Ar—CH), 7.12 (s, Ar—CH), 7.38 (d, J=8 Hz, Ar-2(CH)), 8.00 (d, J=8 Hz,Ar-2(CH)).

EXAMPLE 15 Preparation of compound 46 where R₁, R₂, R₃, and R₄ aremethyl, R₅ is bromo, R′ and R″ are methano, and X=COOH (3-bromo-TTNEB)

The compound was prepared in a manner similar to that of compound 7except that 6-bromo-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalenewas substituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalenein examples 1, 2, 4, and 5. MP: 235° C.; ¹H-NMR (CDCl₃) δ 1.27 (s,2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s, CH₃), 5.40 (s, ═CH), 5.90 (s, ═CH),7.26 (s, Ar—CH), 7.36 (s, Ar—CH), 7.43 (d, J=8 Hz, Ar-2(CH)), 8.04 (d,J=8 Hz, Ar-2(CH)).

EXAMPLE 16 Preparation of compound 47 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ taken together are CH₂—O (epoxide), and X=COOH (TPNED)

The compound was prepared from compound 6 where R₁, R₂, R₃, R₄, R₅ aremethyl. To 1 g (2.76 mmol) of olefin 6 in 5 mL of CH₂Cl₂ was added 600mg (3.46 mmol) of mCPBA and the reaction was stirred at room temperaturefor 2 h. Water was added followed by extraction of the organics withether. The ether layer was washed with water, 1N Na₂CO₃, brine and dried(MgSO₄), filtered and concentrated. Crystallization from MeOH gave thedesired epoxide-methyl ester. The methyl ester was hydrolized inrefluxing methanolic KOH followed by acidification (1N HCl) to give thecrude epoxide-acid 47 which was purified by crystallization fromEtOAc-hex to give 600 mg (1.64 mmol) of a white powder (59% yield). MP:168° C.; ¹H-NMR (CDCl₃) δ 1.26 (s, CH₃), 1.27 (s, CH₃), 1.30 (s, CH₃),1.31 (s, CH₃), 1.69 (s, (2CH₂)), 2.14 (s, CH₃), 3.15 (d, J=5.6 Hz,CH—O), 3.41 (d, J=5.6 Hz, CH-0), 7.09 (s, Ar—CH), 7.28 (d, J=8.3 Hz,Ar-2(CH)), 7.32 (s, Ar—CH), 8.01 (d, J=8.3 Hz, Ar-2(CH)).

EXAMPLE 17 Preparation of compound 48 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ taken together are CH₂—CH₂ (cyclopropyl), and X=COOH(TPNCB)

The compound was prepared from compound 6 where R₁, R₂, R₃, R₄, R₅ aremethyl. To a dry 100 mL three necked round bottom flask fitted with areflux condensor, dropping funnel, and magnetic stir bar was added 722mg (11.65 mmol) of zinc dust, 109 mg (1.105 mmol) of cuprous chloride(CuCl), 7.5 mL of dry THF, and 1.48 g (5.52 mmol) of diiodomethane. Tothe addition funnel is added 1 g (2.76 mmol) of compound 6 in 5 mL ofdry THF. The flask is heated to 80° C., followed by dropwise addition of6. After the addition of 6 was complete, the reaction was allowed toreflux for 30 h or until completion, followed by dilution with 50 mL ofether and 20 mL of saturated aqueous ammonium chloride solution. Theorganic layer was washed with 10% NaOH (3×20 mL), brine and dried overanhydrous MgSO₄. The product was concentrated and purified bypreparative TLC (2% EtOAc-hexane) to give 220 mg (0.59 mmol) of themethyl ester of 48. Hydrolysis of the methyl ester with refluxingmethanolic KOH, followed by acidification (1N HCl), gave 150 mg (0.41mmol) of the desired compound 48 after crystallization from EtOAc-hexane(15% yield). MP: 244° C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 4(CH₃)), 1.39 (s,CH2—CH₂), 1.69 (s, 2(CH₂)), 2.12 (s, CH₃), 6.98 (d, J=8.4 Hz, Ar-2(CH)),7.06 (s, Ar—CH), 7.29 (s, Ar—CH), 7.91 (d, J=8.4 Hz, Ar-2(CH)).

EXAMPLE 18 Preparation of compound 49 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′=H and R″=CH₃, and X=COOH (PTNEB)

The compound was prepared from compound 7 where R₁, R₂, R₃, R₄, R₅ aremethyl. To 1 g (2.87 mmol) of compound 7 in 25 mL of EtOAc was added 10mg of 10% Pd/C. The mixture was degassed under vacuum followed byaddition of H₂, and allowed to stir under H₂ for 2 h. The reaction wasfiltered through celite and the product crystallized from EtOAc-hexaneto give 750 mg (2.14 mmol) of the desired product 49 (75% yield). MP:208° C.; ¹H-NMR (CDCl₃) δ 1.24 (s, CH₃), 1.25 (s, CH₃), 1.26 (s, CH₃),1.29 (s, CH₃), 1.61 (d, J=7.2 Hz, CH₃), 1.67 (s, 2(CH₂)), 2.12 (s, CH₃),4.30(g, J=7.2 Hz, CH), 7.02 (s, Ar—CH), 7.20 (s, Ar—CH), 7.24 (d, J=8.4Hz, Ar-2(CH)), 7.99 (d, J=8.4 Hz, Ar-2(CH)).

EXAMPLE 19 Preparation of compound 50 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=methylidene cyclopentane, and X=COOH (PTNCB)

The compound was prepared from compound 4 and where R₁, R₂, R₃, R₄, R₅are methyl. To 1 g (2.87 mmol) of 4 in 25 mL of THF at 0° C. was added8.6 mL of a 1M cyclopentenyl magnesium chloride solution (8.6 mmol).After stirring for 30 m, water was added and acidified with 5 N HCl. Theacidified mixture was heated for 5 m, cooled, and the organic productextracted with EtOAc. The EtOAc layer was washed with water and brine,dried (MgSO₄), filtered, and concentrated to give the crude product.Crystallization from EtOAc-hexane gave 340 mg (0.85 mmol) of 50 as awhite powder (30% yield). MP: 201° C.; ¹H-NMR (CDCl₃) δ 1.27 (s,4(CH₃)), 1.64 (br t, CH₂), 1.68 (s, 2(CH₂)), 1.70 (br t, CH₂), 1.97 (s,CH₃), 2.15 (br t, CH₂), 2.56 (br t, CH₂), 7.04 (s, Ar—CH), 7.05 (s,Ar—CH), 7.29 (d, J=8 Hz, Ar-2(CH)), 7.97 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 20 Preparation of compound 51 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=isopropylidene, and X=COOH (PTNIB)

The compound was prepared from compound 4 and where R₁, R₂, R₃, R₄, R₅are methyl. To 1 g (2.87 mmol) of 4 in 25 mL of THF at 0° C. was added8.6 mL of a 1M isopropyl magnesium chloride solution (8.6 mmol). Afterstirring for 30 m, water was added and acidified with 5 N HCl. Theacidified mixture was heated for 5 m, cooled, and the organic productextracted with EtOAc. The EtOAc layer was washed with water and brine,dried (MgSO₄), filtered, and concentrated to give the crudeisopropylidene product. Crystallization from EtOAc-hexane gave 550 mg(1.46 mmol) of 51 as a white powder (51% yield). MP: 297° C.; ¹H-NMR(CDCl₃) δ 1.25 (br s, 4(CH₃)), 1.64 (s, ═CCH₃), 1.66 (s, ═CCH₃) 1.87 (s,2(CH₂)), 1.96 (s, CH₃), 7.00 (s, Ar—CH), 7.03 (s, Ar—CH), 7.25 (d, J=8Hz, Ar-2(CH)), 7.97 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 21 Preparation of compound 52 where R₁, R₂, R₃, R₄ and R₅ aremethyl, R′ and R″=oxo, Z=S, and X=COOH (TTNCTC)

To 1 g (4.9 mmol) of1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydro-naphthalene and 1 g (4.9 mmol)of mono methyl thiophene carboxylic acid chloride in 25 mL of CH₂Cl₂ wasadded 1 g (7.5 mmol) of AlCl₃. The reaction was heated to reflux for 15m followed by cooling and addition of 20% aqueous HCl. The product wasextracted with EtOAc, washed (H₂O, brine), dried (MgSO₄), filtered,concentrated, and purified by crystallization from MeOH to give 450 mg(1.21 mmol) of the methyl ester of 52 (25% yield). The methyl ester washydrolized in methanolic KOH followed by acidification (20% HCl)extraction with EtOAc, washed (H₂O, brine), dried (MgSO₄), filtered,concentrated, and purified by crystallization from EtOAc-hexane to give375 mg (1.05 mmol) of 52 (87% yield). MP: 206° C.; ¹H-NMR (CDCl₃) δ 1.26(s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s, 2(CH₂)), 2.38 (s, CH₃), 7.21 (s,Ar—CH), 7.44 (s, Ar—CH), 7.48 (d, J=4 Hz, Thio Ar—CH), 7.85 (d, J=4 Hz,Thio Ar—CH).

EXAMPLE 22 Preparation of compound 53 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=methano, Z=S, and X=COOH (TTNETC)

Compound 53 was prepared from the methyl ester of 52 in a manner similarto examples 4 and 5. MP: 200° C.; ¹H-NMR (CDCl₃) δ 1.26 (s, 2(CH₃)),1.30 (s, 2(CH₃)), 1.69 (s, 2(CH₂)), 2.10 (s, CH₃), 5.21 (s, ═CH), 5.88(s, ═CH), 6.76 (d, J=4 Hz, Thio Ar—CH), 7.11 (s, Ar—CH), 7.23 (s,Ar—CH), 7.68 (d, J=4 Hz, Thio Ar—CH).

EXAMPLE 23 Preparation of compound 54 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=oxo, and X=tetrazole (3-methyl-TTNCBT)

To 500 mg (1.51 mmol) of4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzonitrile(synthesized by AlCl₃ catalyzed condensation of1,1,4,4,6-pentamethyl-1,2,3,4-tetra hydronaphthalene with 4-cyanobenzoicacid chloride in CH₂Cl₂) in toluene was added 342 mg (1.66 mmol) oftrimethyl tin azide. The mixture was refluxed for 23 h and cooled togive 537 mg (1.44 mmol) of the desired tetrazole 54 as a whiteprecipitate (96% yield). LRMS: 374.15; ¹H-NMR (CD₃SOCD₃) δ 1.19 (s,2(CH₃)), 1.32 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 2.25 (s, CH₃), 3.19 (s,N—H), 7.30 (s, Ar—CH), 7.32 (s, Ar—CH), 7.90 (d, J=8 Hz, Ar-2(CH)), 8.20(d, J=8 Hz, Ar-2(CH)).

EXAMPLE 24 Preparation of compound 55 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=methano, and X=tetrazole (3-methyl-TTNEBT)

To 500 mg (1.52 mmol) of4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzonitrile(synthesized by AlCl₃ catalyzed condensation of1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene with 4-cyanobenzoicacid chloride in CH₂Cl₂ followed by treatment of the ketone with CH₃PPh₃Br—NaNH₂) in toluene was added 342 mg (1.67 mmol) of trimethyl tinazide. The mixture was refluxed for 23 h and cooled to give 535 mg (1.44mmol) of the desired tetrazole 55 as a white precipitate (95% yield).LRMS: 372.25; ¹H-NMR (CD₃SOCD₃) δ 1.21 (s, 2(CH₃)), 1.24 (s, 2(CH₃)),1.68 (s, 2(CH₂)), 1.92 (s, CH₃), 2.55 (s, N—H), 5.27 (═CH), 5.97 (s,═CH), 7.10 (s, Ar—CH), 7.18 (s, Ar—CH), 7.47 (d, J=8 Hz, Ar-2(CH)), 8.00(d, J=8 Hz, Ar-2(CH)).

EXAMPLE 25 Preparation of compound 25 where R₁, R₂, R₃, and R₄ aremethyl, R′ and R″=oxo, and X=COOMe

The compound was prepared in a manner similar to that of compound 4except that 1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene wassubstituted for 1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene and4-methyl ester pyridinic 2-acid chloride was substituted for mono-methylterephthalic acid chloride (see examples 1 and 2).

EXAMPLE 26 Preparation of compound 56 where R₁, R₂, R₃, and R₄ aremethyl, R′ and R″=methano, and X=COOH (TTNEP)

Compound 25 was treated with CH₃PPh₃ Br—NaNH₂ as in example 4.Hydrolysis of the resulting olefinic methyl ester with methanolic KOH,followed by acidification (20% HCl) and crystallization fromEtOAc-hexane gave compound 56. MP: 173° C.; ¹H-NMR (CDCl₃) δ 1.26 (s,(CH₃)), 1.27 (S, CH₃), 1.30 (s, 2(CH₃)), 1.70 (s, (CH₂)), 5.70 (s, ═CH),6.10 (s, ═CH), 7.08 (d, J=8 Hz, Pyr-CH), 7.27 (s, Ar—CH), 7.19 (d, J=8Hz, Ar—CH), 7.39 (d, J=8 Hz, Ar—CH), 8.28 (d, J=8 Hz, Pyr-CH), 9.31 (s,Pyr-CH).

EXAMPLE 27 Preparation of compound 57 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=oxo, and X=COOH

Compound 57 was prepared in a manner similar to that of compound 6(example 4) except that 4-methylester-pyridinic-2-acid chloride wassubstituted for mono-methyl terephthalic acid chloride (see examples 1and 2). The resulting methyl ester was hydrolyzed as in example 5 togive compound 57. ¹H-NMR (CDCl₃) δ 1.22 (s, 2(CH₃)), 1.30 (s, 2(CH₃)),1.69 (s, 2(CH₂)), 2.40 (s, CH₃), 7.22 (s, Ar—CH), 7.43 (s, Ar—CH), 8.13(d, J=8.0 Hz, Pyr-CH), 8.54 (d, J=8 Hz, Pyr-CH), 9.34 (s, Pyr-CH).

EXAMPLE 28 Preparation of compound 58 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″=methano, and X=COOH (TPNEP)

The methyl ester from example 26 was treated with CH₃PPh₃ Br—NaNH₂ as inexample 4 followed by hydrolysis with methanolic KOH at reflux for 1 hand acidification with 20% aqueous HCl and crystallization fromEtOAc-hexane to give compound 58. MP: 235° C.; ¹H-NMR (CDCl₃) δ 1.27 (s,2(CH₂)), 1.31 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 2.00 (s, CH₃), 5.55 (s,═CH), 6.57 (s, ═CH), 7.06 (d, J=8.3 Hz, Pyr-CH), 7.12 (s, Ar—CH), 7.14(s, Ar—CH), 8.20 (d, J=8.1 Hz, Pyr-CH). 9.29 (s, Pyr-CH).

EXAMPLE 29 Preparation of methyl 2-acetyl-5-pyridinecarboxylate 32

To a slurry of the 2,5-pyridinedicarboxylic acid 29 (34 g, 0.2 mol) in120 mL of methanol at 0° C. was added dropwise 15 mL of thionyl chlorideand the resulting slurry was warmed up to room temperature, giving riseto a clear solution. The mixture then was heated at reflux for 12 h andto afford a yellow slurry. Filtration of the reaction mixture provideddimethyl-2,5-pyridinedicarboxylate 30 in quantitative yield as a yellowcrystalline solid.

The pyridinedicarboxylate 30 (19.5 g, 0.1 mol) was treated with solidKOH (6.51 g, 0.1 mol) in 300 mL of methanol at room temperature for 2 h,giving rise to a thick pale white suspension, which was filtered anddried to provide the mono-potassium pyridinecarboxylate 31 inquantitative yield.

The crude mono-pyridinecarboxylate 31 (880 mg, 4 mmol) was treated with3 mL of thionyl chloride at reflux for 2 h and the excess SOCl₂ wasremoved by the usual method. To the crude acid chloride in 8 mL of THFat −78° C. was added slowly a freshly prepared 1.0M ether solution (5.5mL, 5.5 mmol) Me₂CuLi. The resulting dark slurry was allowed to stir at−78° C. for 60 min. and then was quenched with 2% HCl. Standard work-upand chromatography of the crude mixture affordedmethyl-2-acetyl-5-pyridine-carboxylate 32 in over 56% yield as a yellowsolid.

EXAMPLE 30 Preparation of compound 58 (TPNEP) (by an alternate schemethan in Example 28) and of corresponding ester Et-58 where R₁, R₂, R₃,R₄, and R₅ are methyl, R′ and R″ are methano, and ═COOH

A solution of 2-bromotoluene (8.5 g, 50 mmol) and2,2-dichloro-2,2-dimethyl-hexane (9.15 g, 50 mmol) in 100 mL ofdichloroethane was treated with aluminum trichloride (0.66 g, 5 mmol).The resulting dark brown solution was allowed to stir at roomtemperature for 30 min. and was then quenched with ice. Removal ofsolvent and recrystallization from methanol afforded2-bromo-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene 33 in 95%yield as a white solid. A THF (4 mL) solution containing thebromocompound 33 (141 mg, 0.5 mmol) at −78° C. was treated with a 1.6 Mhexane solution (0.4 mL, 0.6 mmol) of n-BuLi, and the resulting mixturewas then cannulated to a THF (2 mL) solution of the2-acetyl-5-pyridinecarboxylate 32 (72 mg, 0.4 mmol) at −78° C. Themixture was allowed to stir at −78° C. for 60 min. and was quenched with2% HCl. Removal of the solvent and chromatography of the crude mixtureprovided the intermediate 34, which was then treated with 5% HCl atreflux followed by KOH-MeOH at 70° C. for 30 min. Standard work-up andchromatography of the crude mixture provided2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2naphthyl)ethenyl]pyridine-5-carboxylic acid 58 in over 50% yield as awhite solid. 1H-NMR (CDCl₃) δ 1.27 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.70(s, 2(CH₂)), 2.00 (s, CH₃), 5.56 (s, ═CH), 6.55 (s, ═CH), 7.08 (d, J=8.3Hz, Pyr-CH), 7.12 (s, Ar—CH), 7.15 (s, Ar—CH), 8.23 (d, J=8.3 Hz,Pyr-CH) and 9.32 (s, Pyr-CH).

Treatment of the pyridinecarboxylic acid 58 (15 mg, 0.004 mmol) with onedrop of SOCl₂ in 5 mL of ethanol at reflux for 60 m, followed by a flashchromatography, gave rise to a quantitative yield of the ethyl esterEt-58 as a white solid. ¹H-NMR (CDCl₃) δ 1.27 (s, 2(CH₃)), 1.31 (s,2(CH₃)), 1.40 (t, J=7.1 Hz, —CH₂CH₃), 1.70 (s, 2(CH₂)), 1.99 (s, CH₃),4.40(q, J=7.1 Hz, —CH₂CH₃), 5.51 (s, ═CH), 6.53 (s, ═CH), 7.01 (d, J=8.0Hz, Pyr-CH), 7.12 (s, Ar—CH), 7.14 (s, Ar—CH), 8.15 (d, J=8.0 Hz,Pyr-CH) and 9.23 (s, Pyr-CH).

EXAMPLE 31 Preparation of 3-acetyl-2-pyridinecarboxylic acidN,N-diisopropylamide 36a

The mono-potassium pyridinecarboxylate 31 (1.1 g, 5 mmol) was treatedwith SOCl₂ (5 mL, excess) at 70° C. for 2 h and the excess thionylchloride was removed to give a yellowish solid. To a solution ofdiisopropylamine (1 g, 10 mmol) in 10 mL of methylene chloride at 0° C.was added the CH₂Cl₂ solution (10 mL) of the above acid chloride. Theresulting slurry was allowed to stir at room temperature for 3 h and wasfiltered from the ammonium salts. Removal of solvent and chromatographyof the crude residue afforded the product 36a in 90% yield as a whitesolid.

EXAMPLE 32 Preparation of compounds 60 (TPNEPC) and 61 (3TTNEPE)

2-Bromo-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene 33 (620 mg,2.2 mmol) and the acetylpyridineamide 36a (500 mg, 2 mmol) wereconverted by a similar method as described above to obtain theintermediate 34 in over 80% yield. To a solution of the pyridine amide34 (432 mg, 1 mmol) in 5 mL of THF at −78° C. was added 1.5 M DIBALtoluene solution (0.7 mL, 1.05 mmol) and the resulting yellow clearsolution was warmed up to −20° C. slowly in 60 min. and then wasquenched with water. Removal of solvent and chromatography of the crudemixture afforded the pyridinealdehyde 35 in 83% yield as a white solid.¹H-NMR (CDCl₃) δ 1.25 (s, 2(CH₃)), 1.29 (s, 2(CH₃)), 1.70 (s, 2(CH₂)),1.96 (s, CH₃), 5.47 (s, ═CH), 5.92 (s, ═CH), 7.10 (s, Ar—CH), 7.11 (s,Ar—CH), 7.70 (d, J=8.0 Hz, Pyr-CH), 7.88 (d, J=8.0 Hz, Pyr-CH), 8.72 (s,Pyr-CH), 10.06 (s, CHO).

The pyridinealdehyde 35 (10 mg, 0.03 mmol) was treated with 2.0 mL ofH₂O₂ in 2 mL of methanol-water 1:1 mixture at room temperature for 10 hand then was quenched with 10% HCl. Extraction of the mixture with EtOAc(40 mL) and removal of solvent gave rise to5-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-2-carboxylicacid 60 as a white solid in almost quantitative yield. ¹H-NMR (CDCl₃) δ1.26 (s, 2(CH₃)), 1.30 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 1.94 (s, CH₃),5.47 (s, ═CH), 5.92 (s, ═CH), 7.10 (s, Ar-2(CH)), 7.76 (bs, Pyr-CH, 8.16(bs, Pyr-CH) and 8.65 (s, Pyr-CH). Treatment of the pyridinecarboxylicacid 60 (5 mg) with one drop of SOCl₂ in 1 mL of ethanol at reflux for60 min followed by a flash chromatography gave rise to a quantitativeyield of the ethyl ester 61 as a white solid. ¹H-NMR (CDCl₃) δ 1.26 (s,2(CH₃)), 1.29 (s, 2(CH₃)), 1.44 (t, J=7.1 Hz, —CH₂ CH₃), 1.69 (s,2(CH₂)), 1.95 (s, CH₃), 4.46 (q, J=7.1 Hz, —CH₂CH₃)), 5.43 (s, ═CH),5.88 (s, ═CH), 7.09 (s, Ar—CH), 7.10 (s, Ar—CH), 7.64 (d, J=8.0 Hz,Pyr-CH), 8.03 (d, J=8.0 Hz, Pyr-CH) and 8.68 (s, Pyr-CH).

EXAMPLE 33 Preparation of compound 62 where R₁, R₂, R₃, R₄, R₅, aremethyl and R′ and R″ together are CH₂CH₂ (TPNCP)

To 162 mg (2.48 mmol) of zinc dust, 25 mg (0.25 mmol) of CuCl, and 332mg (1.24 mmol) of CH₂I₂ in 3 mL of dry ether was added dropwise 150 mg(0.413 mmol) of olefin 26 where R₁, R₂, R₃, R₄, R₅ are methyl in 5 mL ofdry ether. The mixture was heated at reflux for 12 h or until completeby H-NMR. Water was added and the organics extracted with ether, washedwith NH₄Cl, brine and dried over MgSO₄. The desired cyclopropyl compoundwas purified by crystallization from ether-MeOH to give 60 mg (0.159mmol) of the methyl ester of 62 as a pale yellow solid (39% yield). MP:177° C.; ¹H-NMR (CDCl₃) δ 1.27 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.35 (s,CH₂), 1.70 (s, 2(CH₂)), 1.85 (s, CH₂), 2.11 (s, CH₃), 3.90 (s, CH₃),6.75 (d, J=8.0 Hz, Pyr-CH), 7.14 (s, Ar—CH), 7.26 (s, Ar—CH), 7.98 (d,J=8.0 Hz, Pyr-CH) and 9.23 (s, Pyr-CH).

To 60 mg (0.16 mmol) of the above methyl ester in 10 mL of MeOH wasadded 1 mL of an aqueous 6N KOH solution. After stirring at roomtemperature for 1 h, the hydrolysis was complete and the reaction wasacidified with 1 N aqueous HCl until the solids precipitated. Theproduct was extracted with ether, washed with water, brine and driedover MgSO₄. Crystallization from EtOAc-hexanes gave 33 mg (0.094 mmol)of the pyridinal carboxylic acid 62 (59% yield). MP: 275° C.;¹H-NMR(CDCl₃) δ 1.25 (s, 2(CH₃)), 1.35 (s, 2(CH₃)), 1.40 (s, CH₂), 1.72(s, 2(CH₂)), 1.85 (s, CH₂), 2.15 (s, CH₃), 6.78 (d, J=8.0 Hz, Pyr-CH),7.14 (s, Ar—CH), 7.26 (s, Ar—CH), 8.02 (d, J=8.0 Hz, Pyr-CH) and 9.15(s, Pyr-CH).

EXAMPLE 34 Preparation of compound 63 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, X=CONHR₉, and R₉=4-hydroxyphenyl(3-methyl-TTNEHBP)

To 750 mg (10 mmol) of DMF in 22 mL of anhydrous ether was added 1.3 g(10 mmol) of oxalyl chloride. The reaction was stirred for 1 h, followedby removal of solvent to give a crude white solid(dimethylchloroformadinium chloride). To the dimethylchloroformadiniumchloride was added 2.87 g (8.24 mmol) of compound 7 in 12 mL of dry DMF.The reaction was stirred for 20 m at room temperature followed bycooling to 0° C. The cooled solution of the acid chloride of 7 was addeddropwise to a cooled DMF (0° C.) solution containing 3.62 g (33 mmol) of4-aminophenol and 1.68 g (16.3 mmol) of triethyl amine. After stirringat 0° C. for 30 m, the reaction was warmed to room temperature for 12 h.Aqueous 20% HCl was added and the resulting solid was filtered andwashed with water, acetone, and EtOAc to give 600 mg (1.36 mmol) of thedesired compound 63 (17% yield). ¹H-NMR (CDCl₃) δ 1.29 (s, 2(CH₃)), 1.31(s, 2(CH₃)), 1.71 (s, (CH₂)), 1.99 (s, CH₃), 5.31 (s, ═CH), 5.80 (s,═CH), 6.85 (d, Ar-2(CH)), 7.09 (s, Ar—CH), 7.16 (s, Ar—CH), 7.40 (d,Ar-2(CH)), 7.48 (d, Ar-2(CH)), 8.40 (d, Ar-2(CH)).

EXAMPLE 35 Preparation of compound 64 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, X=CONHR₉ and R₉=4-fluorophenyl(3-methyl-TTNEFBP)

The compound was prepared in a manner similar to that of compound 63except that 4-fluoroaniline was substituted for 4-aminophenol. MP: 203°C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.70 (s,2(CH₃)), 1.96 (s, CH₃), 5.33 (s, ═CH), 5.81 (s, ═CH), 7.05 (d, J=9 Hz),Ar-2(CH)), 7.09 (s, Ar—CH), 7.13 (s, Ar—CH), 7.39 (d, J=8.4 Hz,Ar-2(CH)), 7.59 (dd, J=5,9 Hz, Ar-2CH), 7.75 (br s NH), 7.78 (d, J=8.4Hz, Ar-2(CH)).

EXAMPLE 36 Preparation of compound 65 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, X=CONHR₉, and R₉=4-phenylcarboxylic acid(3-methyl-TTNECBP)

The compound was prepared in a manner similar to that of compound 63except that methyl 4-aminophenyl carboxylate was substituted for4-aminophenol. The resulting ester was hydrolyzed in methanolic KOH,followed by acidification (20% HCl) to give the desired compound 65. MP:200° C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s,2(CH₂)), 1.97 (s, CH₃), 5.34 (s, ═CH), 5.85 (s, ═CH), 7.09 (s, Ar—CH),7.14 (s, Ar—CH), 7.40 (d, J=8 Hz, Ar—CH), 7.80 (d, J=8 Hz, Ar-2(CH)),7.87 (br s, Ar-2(CH)), 8.14 (br s, Ar-2(CH)).

EXAMPLE 37 Preparation of compound 66 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are oxo, X=CONHR₉, and R₉=3-hydroxyphenyl(3-methyl-m-TTNCHBP)

To 750 mg (10 mml) of DMF in 22 mL of anhydrous ether was added 1.3 g(10 mmol) of oxalyl chloride. The reaction was stirred for 1 h, followedby removal of solvent to give a crude white solid(dimethylchloroformadinium chloride). To the dimethylchloro-formadiniumchloride was added 2.88 g (8.24 mmol) of compound 4 in 12 mL of dry DMF.The reaction was stirred for 20 m at room temperature, followed bycooling to 0° C. The cooled solution of the acid chloride of 7 was addeddropwise to a cooled DMF (0° C.) solution containing 3.62 g (33 mmol) of4-aminophenol and 1.68 g (16.3 mmol) of triethyl amine. After stirringat 0° C. for 30 m, the reaction was warmed to room temperature for 12 h.Aqueous 20% HCl was added and the resulting solid was filtered andwashed with water, acetone, and EtOAc to give 750 mg (1.70 mmol) of thedesired compound 66 (21% yield). MP: 182° C.; ¹H-NMR (CDCl₃) δ 1.22 (s,2(CH₃)), 1.32 (s, 2(CH₃)), 1.70 (s, 2(CH₂)), 2.37 (s, CH₃), 6.58 (m,Ar-2(CH)), 7.20 (d, J=8 Hz, Ar—CH), 7.22 (s, Ar—CH), 7.28 (s, Ar—Ch),7.91 (d, J=8.3 Hz, Ar-2(CH)), 8.26 (d, J=8.3 Hz, Ar-2(CH)).

EXAMPLE 38 Preparation of compound 67 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, X=CONHR₉, and R₉=3-hydroxyphenyl(3-methyl-m-TTNEHBP)

The compound was prepared in a manner similar to that of compound 63except that 3-aminophenol was substituted for 4-aminophenol. MP: 136°C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.70 (s,2(CH₂)), 1.97 (s, CH₃), 5.35 (s, ═CH), 5.84 (s, ═CH), 6.57 (m,Ar-2(CH)), 7.09 (s, Ar—CH), 7.14 (s, Ar—CH), 7.16 (m, Ar—CH), 7.39 (d,J=8.3 Hz, Ar-2(CH)), 8.09 (d, J=8.3 Hz, Ar-2(CH)).

EXAMPLE 39 Preparation of compound 68 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, X=CONHR₉, and R₉=2-hydroxyphenyl(3-methyl-o-TTNCHBP)

The compound was prepared in a manner similar to that of compound 63except that 2-aminophenol was substituted for 4-aminophenol. MP: 180°C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s,2(CH₂)), 1.97 (s, CH₃), 5.35 (s, ═CH), 5.84 (s, ═CH), 6.9 (m, Ar—CH),7.08-7.2 (m, Ar—CH), 7.09 (s, Ar—CH), 7.13 (s, Ar—CH), 7.42 (d, J=8.4Hz, Ar-2(CH)), 7.83 (d, J=8.4 Hz, Ar-2(CH)), 8.03 (brs, Ar—CH), 8.64 (s,NH).

EXAMPLE 40 Preparation of compound 69 where R₁, R₂, R₃, R₄, AND R₅ aremethyl, R′ and R″ are methano, X=CONHR₉, and R₉=3-phenylcarboxylic acid(3-methyl-m-TTNECBP)

The compound was prepared in a manner similar to that of compound 63except that methyl-3-amino phenyl carboxylate was substituted for4-aminophenol. The resulting ester was hydrolyzed in methanolic KOHfollowed by acidification (20% HCl) to give the desired compound 69. MP:250° C.; ¹H-NMR (CDCl₃) δ 1.28 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.71 (s,2(CH₂)), 1.97 (s, CH₃), 5.34 (s, ═CH), 5.85 (s, ═CH), 7.09 (s, Ar—CH),7.14 (s, Ar—CH), 7.40 (d, J=8 Hz, Ar-2(CH)), 7.55 (m, Ar—CH), 7.76 (m,Ar—CH), 7.80 (d, J=8 Hz, Ar-2(CH)), 7.87 (s, Ar—CH), 8.14 (s, NH).

EXAMPLE 41 Preparation of compound 70 where R₁, R₂, R₃, R₄, and R₅ aremethyl, R′ and R″ are methano, n=0, and X=COOH

The compound was prepared in a manner similar to that of compound 7except that 1,1,3,3,5-pentamethylindane was substituted for1,1,4,4,6-pentamethyl-1,2,3,4-tetrahydronaphthalene in examples 1, 2, 4,and 5. MP: 145° C.; ¹H-NMR (CDCl₃) δ 1.05 (s, 2(CH₃)), 1.28 (s, CH₃),1.31 (s, CH₃), 1.38 (s, CH₂), 1.98 (s, CH₃), 5.34 (s, CH), 5.84 (s, CH),6.90 (s, Ar—CH), 6.92 (s, Ar—CH), 7.36 (d, J=8.4 Hz, Ar-2(CH)), 8.00 (d,J=8.4 Hz, Ar-2(CH)).

EXAMPLE 42 Preparation of compound 71 where R₁, R₂, R₃, R₄, R₅, and R₁₄are methyl, R′ and R″ are methano, n=0, and X=COOH

The compound was prepared in a manner similar to that of compound 7except that 1,1,2,3,3,5-pentamethylindane was substituted for1,1,4,4,6-pentamethyl-1,2,3,4 tetrahydronaphthalene in examples 1, 2, 4,and 5. MP: 217° C.; ¹H-NMR (CDCl₃) δ 1.01 (d, J=7.3 Hz, CH₃), 1.08 (s,CH₃), 1.10 (s, CH₃), 1.27 (s, CH₃), 1.30 (s, CH₃), 1.88 (q, CH), 2.00(s, CH₃), 5.35 (s, ═CH), 5.85 (s, ═CH), 6.95 (s, Ar—CH), 6.98 (s,Ar—CH), 7.38(d, J=8.3 Hz, Ar-2(CH)), 8.00 (d, J=8.3 Hz, Ar-2(CH)).

EXAMPLE 43 Preparation of compound 72 where R₁, R₂, R₃, R₄, and R₅, aremethyl, R′ and R″ are H, and X=COOH

The compound was prepared in a manner similar to that of compound 4(examples 1 and 2) except that methyl-4-(bromomethyl)benzoate wassubstituted for mono-methyl terephthalic acid chloride. MP: 237° C.;¹H-NMR (CDCl₃) δ 1.23 (s, 2(CH₃)), 1.27 (s, 2(CH₃)), 1.67 (s, 2(CH₂)),2.16 (s, CH₃), 4.06 (s, CH₂), 7.01 (s, Ar—CH), 7.08 (s, Ar—CH), 7.25 (d,J=8 Hz, Ar-2(CH)), 8.01 (d, J=8 Hz, Ar-2(CH)).

EXAMPLE 44 Preparation of compound 111 where R₁, R₂, R₃, R₄, R₅ aremethyl and R′ and R″ together are CH₂CH₂

To 200 mg (0.573 mmol) of ester 25 in 10 mL of dry dichloro ethane underdry nitrogen at 0° C. was added 0.29 mL (2.87 mM) of Et₂Zn. To thissolution was added ClCH₂I dropwise via a syringe and the reactionmixture was stirred by 0° C. for 10 m. The solution was then warmed to55° C. for 6 h or until complete by TLC. Water was added and theorganics extracted with ether, washed with NH₄Cl, brine and dried overMgSO₄. The desired cyclopropyl compound was purified by SiO₂ columnchromatography to give 30 mg (0.083 mmol) of the methyl ester of 73 as awhite solid (14% yield). MP: 160° C.; ¹H-NMR (CDCl₃) δ 1.26 (s, 2(CH₃)),1.3 (s, 2(CH₃)), 1.35 (s, CH₂)), 1.70 (s, 2(CH₂)), 1.72 (S, CH₂), 3.90(S, OCH₃), 6.87 (d, J=8.0 Hz, Pyr-CH), 7.12 (d, J=8 Hz, Ar—CH), 7.27 (s,Ar—CH), 7.32 (d, J=8 Hz, Ar—CH), 7.98 (d, J=8.0 Hz, Pyr-CH) and 9.08 (s,Pyr-CH).

To 30 mg (0.083 mmol) of the above methyl ester in 5 mL of MeOH wasadded 1 mL of an aqueous 6N KOH solution. After stirring at RT for 1 h,the hydrolysis was complete and the reaction was acidified with 1 Naqueous HCl until the solids precipitated. The product was extractedwith ether, washed with water, brine and dried over MgSO₄.Crystallization from EtOAc-hexanes gave 18 mg (0.051 mmol) of thepyridinal carboxylic acid 111 (62% yield). MP: 255° C.; ¹H-NMR(CDCl₃) δ1.25 (s, 2(CH₃)), 1.31 (s, 2(CH₃)), 1.40 (s, CH₂), 1.72 (s, 2(CH₂)),1.75 (s, CH₂), 6.89 (d, J=8.0 Hz, Pyr-CH), 7.12 (d, J=8 Hz, Ar—CH), 7.27(s, Ar—CH), 7.30 (d, J=8 Hz, Ar—CH), 8.02 (d, J=8.0 Hz, Pyr-CH), and9.11 (s, Pyr-CH).

EXAMPLE 45 Preparation of compound 112, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ together are oxime (HO—N═); X=COOH (oxime of3-methyl-TTNCB)

3-methyl-TTNCB (4.41 g, 12.6 mmol) in EtOH (10 mL) and pyridine (15.3mL) was treated with hydroxylamine hydrochloride (4.38 g, 63 mmol), andthe mixture was heated at reflux. After 6 h, the mixture was cooled toroom temperature and the ethanol was removed in vacuo. The residue wastaken-up in water and the aqueous layer was adjusted to pH=4-5 with 1 Maqueous HCl. The aqueous solution was extracted 3 times with EtOAc; theorganic layers were combined, and washed with water (2×) and brine. Theorganic solution was dried (NaSO₄), filtered, and concentrated to give afoamy white solid. Recrystallization (CH₂Cl₂/ether/hexanes) gave a whitesolid, 4.05 g (88%). MP: 204-209° C. (d); HRMS: 366.2060 (MH⁺); ¹H-NMR(CDCl₃/d-4 MeOH) δ 1.22 (s, 6H, 2 (CH₃)), 1.32 (s, 6H, 2(CH₃)), 1.69 (s,4H, 2(CH₂)), 2.11 (s, 3H, CH₃,), 6.99 (s, 1H, Ar—CH), 7.20 (s, 1H,Ar—CH), 7.53 (½ABq, 2H, J=8.4 Hz, Δv=183.0 Hz, Ar—CH), 7.99 (½ABq, 2H,J=8.4 Hz, Δv=183.0 Hz-Ar—CH).

EXAMPLE 46 Preparation of compound 113, where R₁, R₂, R₃, R₄ are methyl;R₅ is bromo; R′ and R″ are oxime (HO—N═); X=COOH (oxime of3-bromo-TTNCB, 41)

3-bromo-TTNCB methyl ester (399 mg, 0.93 mmol) in MeOH (2 mL) wastreated with hydroxylamine hydrochloride (97 mg. 1.4 mmol) and KOH (156mg, 2.8 mmol), and the mixture was heated at reflux for 3 h. Thereaction was worked-up in a manner identical to that described for 112to give a white solid 330 mg (83%). MP: 240-244° C. (d); ¹H-NMR (CDCl₃)δ 1.26 (s, 6H, 2(CH₃)), 1.33 (s, 6H, 2(CH₃)), 1.72 (s, 4H, 2(CH₂)), 7.30(s, 1H, Ar—CH), 7.54 (s, 1H, Ar—CH), 7.92 (½ABq, 2H, J=8.3 Hz Δv=113.5Hz, Ar—CH), 8.20 (½ABq, 2H, J=8.3 Hz Δv=113.5 Hz, Ar—CH).

EXAMPLE 47 Preparation of compound 114, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ are oxime (HO—N═); X=COOH; Z=N; Z′, Z″, Z′″=CH (oximeof 57)

The compound was prepared in a manner similar to that described for 113,except compound 57, rather than 3-methyl-TTNCB, was the starting ketone.Flash chromatography on SiO₂, (hexanes:EtOAc;CHCl₂:isopropanol=10:5:1:1)of the crude product, gave a sticky, white solid, 83 mg (86%). mP:167-172° C. (d); HRMS: 367.2025(MH⁺); FTIR (neat) 3600-3230 (br), 2962,2928, 2664, 1697, 1593, 1296, 1273, 1122, 1028, 964 cm⁻¹, ¹H-NMR(CDCl₃), δ 1.23 (s, 6H, 2 (CH₃)), 1.32 (s, 6H, 2(CH₃)) 169 (s, 4H, 2(CH₂)), 2.14 (s, 6H, CH₃), 7.05 (s, 1H, Ar—CH), 7.22 (s, 1H, Ar—CH),7.49 (½ABq, 2H, J=28.0 Hz, Ar—CH), 8.25 (Y2ABq, 2H, J=8.0 Hz, Ar—CH);9.21 (s, 1H, Ar—CH).

EXAMPLE 48 Preparation of compound 115, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ are methoxyoxime (CH₃O—N═); X=COOH (Methozyozime of3-methyl-TTNCB)

3-methyl-TTNCB (560 mg, 1.60 mmol) in EtOH (2 mL) and pyridine (1.3 mL)was treated with methoxylamine hydrochloride (402 g, 4.81 mmol), and themixture was heated at reflux. After 6 h, the mixture was cooled to roomtemperature and the ethanol was removed in vacuo. The residue wastaken-up in water and the aqueous layer was adjusted to pH=4-5 with 1 Maqueous HCl. The aqueous solution was extracted 3 times with EtOAc, theorganic layers were combined, and washed with water (2×) and brine. Theorganic solution was dried (NaSO₄), filtered, concentrated, andcrystallized (CH₂Cl₂/hexanes) to give a white solid, 564 mg (93%).MP:228-228.5° C.; LRMS: 380 (MH⁺); ¹H-NMR (CDCl₃) δ 1.22 (s, 6H, 2(CH₃)), 1.31 (s, 6H, 2 (CH₃)), 1.69 (s, 4H, 2(CH₂)), 2.08 (s, 3H, CH₃),4.01 (s, 3H, OCH₃), 6.95 (s, 1H, Ar—CH), 7.18 (s, 1H, Ar—CH), 7.57(½ABq, 2H, J=8.4 Hz, Δv=188.3 Hz, Ar—CH), 8.04 (½ABq, 2H, J=8.4 Hz,Δv=188.3 Hz, Ar—CH).

EXAMPLE 49 Preparation of compound 116, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ are methyloxime (CH₃O—N═); X=COOH, Z=N; Z′, Z″, Z′″=CH(methyloxime of 57)

3-methyl-TTNCB methyl ester (151 mg, 0.41 mmol) in EtOH (1 mL) wastreated with methoxylamine hydrochloride (52 mg, 0.62 mmol) and pyridine(70 μL, 0.82 mmol), and the mixture was heated at reflux for 5 h. Thereaction was worked-up in a manner identical to that described for 115to give a solid (169 mg). The crude product was hydrolyzed in excessKOH/MeOH at ambient temperature for 24 h. The methanol was removed invacuo. The residue was taken-up in water and the aqueous layer wasadjusted to pH=4-5 with 1 M aqueous HCl. The aqueous solution wasextracted 3 times with EtOAc; the organic layers were combined, andwashed with water (2×) and brine. The organic solution was dried(NaSO₄), filtered, concentrated, and crystallized (Et₂O/hexanes) to givea white solid, 96 mg (61%). MP: 260-263° C.; ¹H-NMR (CDCl₃) δ 1.22 (s,6H, 2(CH₃)), 1.30 (s, 6H, 2(CH₃)), 1.68 (s, 4H, 2(CH₂)), 2.10 (s, 3H,CH₃), 4.08 (s, 3H, OCH₃), 6.99 (s, 1H, Ar—CH), 7.18 (s, 1H, Ar—CH), 7.63(d, 1H, Py-CH, J=8.0 Hz), 8.31 (d, 1H, Py-CH, J=8.0 Hz), 9.31 (s, 1H,Py-CH).

EXAMPLE 50 Preparation of compound 138, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ are n-butyloxime (n-BuO-N═); X=COOH (n-butylozime of3-methyl-TTNCB)

A solution of the oxime of 3-methyl-TTNCB (compound 112, 121 mg, 0.33mmol) in THF (0.3 mL) and DMPU (0.3 mL) was added at 0° C. to asuspension of NaH (24 mg, 1.0 mmol) in THF (1.0 mL). The suspension wasallowed to warm to room temperature with stirring over 30 minutes, thena solution of n-butyl bromide (136 mg, 110 μL, 1.0 mmol) in THF (1.0 mL)was added. The solution was allowed to warm to room temperature andstirred for 15 hr. Aqueous, saturated NH₄Cl (3.0 mL) was added and theaqueous layer was adjusted to pH=4-5 with 1 M aqueous HCl. The aqueoussolution was extracted 3 times with EtOAc; the organic layers werecombined, and washed with water (2×) and brine. The organic solution wasdried (MgSO₄), filtered, concentrated, and crystallized (ether/hexanes)to give a white solid, 82 mg (58%). MP: 195-198° C.; ¹H-NMR (CDCl₃) δ0.94 (t, 3H, CH₃, J=7.4 Hz), 1.22 (s, 6H, 2(CH₃)), 1.31 (s, 6H, 2(CH₃)),1.69 (s, 4H, 2(CH₂)), 1.72 (mult, 2H, CH₂, J=7.3 Hz), 2.05 (s, 3H, CH₃),4.16 (t, 3H, OCH₂, J=6.7 Hz), 6.97 (s, 1H, Ar—CH), 7.16 (s, 1H, Ar—CH),7.57 (d, 2H, Ar—CHz, J=8.4 Hz), 8.04 (d, 2H, Ar—CH₂, J=8.4 Hz).

EXAMPLE 51 Preparation of compound 139, where R₁, R₂, R₃, R₄, R₅ aremethyl; R′ and R″ are n-propyloxime (n-ProO-N═); X=COOH (n-propyloximeof 3-methyl-TTNCB)

A solution of the oxime of 3-methyl-TTNCB (compound number 112, 121 mg,0.33 mmol) in THF (0.3 mL) and DMPU (0.3 mL) was added at 0° C. to asuspension of NaH (24 mg, 1.0 mmol) in THF (1.0 mL). The suspension wasallowed to warm to room temperature with stirring over 30 minutes, thena solution of n-propyl bromide (122 mg, 90 μL, 1.0 mmol) was added. Thesolution was allowed to warm to room temperature and stirred for 15 hr.Aqueous, saturated NH₄Cl (5.0 mL) was added and the aqueous layer wasadjusted to pH=4-5 with 1 M aqueous HCl. The aqueous solution wasextracted 3 times with EtOAc; the organic layers were combined, andwashed with water (2×) and brine. The organic solution was dried(MgSO₄), filtered, concentrated, and crystallized (ether/hexanes) togive a white solid, 99 mg (73%). MP: 178-180° C.; ¹H-NMR (CDCl₃) δ 0.93(t, 3H, CH₃, J=7.4 Hz), 1.26 (s, 6H, 2(CH₃)), 1.35 (s, 6H, 2(CH₃)), 1.51(mult, 2H, CH₂, J=7.4 Hz), 1.73 (mult, 6H, 2(CH₂)/CH2), 2.09 (s, 3H,CH₃), 4.24 (t, 3H, OCH₂ J=7.4 Hz), 7.00 (s, 1H, Ar—CH), 7.19 (s, 1H,Ar—CH), 7.60 (d, 2H, Ar—CH₂, J=8.5 Hz), 8.07 (d, 2H, Ar—CH₂, J=8.5 Hz).

EXAMPLE 52 Preparation of compound 140, where R₁, R₂, R₃, R₄, and R₅ aremethyl; R′ and R″ are cyanoimine (═N—CN); X=COOH (cyanoimine of3-methyl-TTNCB)

A solution of 3-methyl-TTNCB (350 mg, 1.0 mmol) in methylene chloride(1.5 mL) was treated with bis(trimethylsilyl)carbodiimide (186 mg, 230μL, 1.0 mmol) at room temperature. The solution was cooled to 0° C., andtreated with a 1M solution of TiCl₄ in methylene chloride (2 eq., 2 mL,2 mmol). The resulting dark red solution was heated at reflux for 8 hr.An additional equivalent of TiCl₄ (1 mL of 1M methylene chloridesolution) was added and the reflux was continued for 3 hours, and TLCanalysis indicated complete conversion to one product. The solution wascooled to room temperature, then poured into ice cold aqueous NaHSO₄.The mixture was diluted with ethyl acetate and filtered through a pad ofcelite. The aqueous layer was washed twice with ethyl acetate and theorganic layers were combined. The organic solution was washed twice withwater and once with saturated aqueous NaCl solution, dried (Na₂SO₄),filtered, and concentrated. The residue was crystallized from solutionwith methylene chloride/hexanes/benzene to give a pale yellow solid, 207mg (55%). MP: 217-219° C.; IR (KBr pellet) 3500-2600 br, 2961, 2924,2666, 2210, 1693, 1582, 1560, 1427, 1408, 1314, cm⁻¹; ¹H-NMR (CDCl₃) δ1.26 (s, 6H, 2(CH₃)), 1.32 (s, 6H, 2(CH₃)), 1.72 (s, 4H, 2(CH₂)), 2.11(s, 3H, CH₃), 7.14 (s, 1H, Ar—CH), 7.24 (s, 1H, Ar—CH), 7.90 (d, 2H,Ar—CH, J=8.1 Hz), 8.17 (d, 2H, Ar—CH, J=8.1 Hz); ¹³C-NMR (CDCl₃) δ191.3, 170.5, 148.6, 143.1, 140.4, 133.9, 132.7, 131.8, 130.4, 129.2,125.9, 34.8, 34.7, 34.4, 34.1, 31.7, 31.6, 19.5; FAB MS C₂₄H₂₆N₂O₂: 375(MH⁺).

EXAMPLE 53 Preparation of compound 141, where R₁, R₂, R₃, R₄, and R₅ aremethyl; R′ and R″ are allyloxime (allyl-O—N═); X=COOH (allyloxime of3-methyl-TTNCB)

A solution of the oxime of 3-methyl-TTNCB (compound number 112, 100 mg,0.27 mmol) in THF (0.3 mL) and DMPU (0.3 mL) was added at 0° C. to asuspension of NaH (20 mg, 0.82 mmol) in THF (1.0 mL). The suspension wasallowed to warm to room temperature with stirring over 30 minutes, thena solution of allyl bromide (71 μL, 0.82 mmol) was added. The solutionwas stirred at room temperature for an additional 12 hr. Aqueous,saturated NH₄Cl (5.0 mL) was added and the aqueous layer was adjusted topH=4-5 with 1 M aqueous HCl. The aqueous solution was extracted 3 timeswith EtOAc; the organic layers were combined, and washed with water (2×)and brine. The organic solution was dried (MgSO₄), filtered,concentrated, and crystallized (ether/hexanes) to give a white solid, 87mg (78%). ¹H-NMR (CDCl₃) δ 1.21 (s, 6H, 2(CH₃)), 1.31 (s, 6H, 2(CH₃)),1.69 (s, 4H, 2(CH₂)), 2.06 (s, 3H, CH₃), 4.71 (d, 2H, OCH₂, J=5.6 Hz),5.19 (dd, 1H, ═CH_(trans), J=10.4, 1.5 Hz), 5.27 (dd, 1H, ═CH_(cis),J=17.2, 1.5 Hz), 6.02 (mult, 1H, ═CH), 6.97 (s, 1H, Ar—CH), 7.16 (s, 1H,Ar—CH), 7.57 (d, 2H, Ar—CH, J=8.2 Hz), 8.03 (d, 2H, Ar—CH, J=8.2 Hz).

EXAMPLE 54 Preparation of compound 142, where R₁, R₂, R₃, R₄, and R₅ aremethyl; R′ and R″ are 4-(3-methyl-butenyl-1-carboxy)oxime(HOOC—C═C(CH₃)CH₂—N═); X=COOH (4-(3-methyl-butenyl-1-carboxy)oxime of3-methyl-TTNCB)

A solution of the oxime of 3-methyl-TTNCB (compound number 112, 202 mg,0.55 mmol) in THF (0.5 mL) and DMPU (0.5 mL) was added at 0° C. to asuspension of NaH (38 mg, 1.7 mmol) in THF (1.7 mL). The suspension wasallowed to warm to room temperature with stirring over 30 minutes, thena solution of ethyl 4-bromo-3-methyl-but-2-ene carboxylate (343 mg, 1.7mmol) was added. The solution was allowed to warm to room temperatureand stirred for 12 hr. Aqueous, saturated NH₄Cl (5.0 mL) was added andthe aqueous layer was adjusted to pH=4.5 with 1 M aqueous HCl. Theaqueous solution was extracted 3 times with EtOAc; the organic layerswere combined, and washed with water (2×) and brine. The organicsolution was dried (MgSO₄), filtered, and concentrated to give a yellowoil. Radial chromatography (1 mm SiO₂ plate, 9:1=hexane; EtOAc withgradual addition of 2% isopropanol) gave a yellow oil. The ester washydrolyzed in excess KOH/MeOH at room temperature for 24 hr. Themethanol was removed in vacuo. The residue was taken-up in water and theaqueous layer was adjusted to pH=4-5 with 1 M aqueous HCl. The aqueoussolution was extracted 3 times with EtOAc; the organic layers werecombined, and washed with water (2×) and brine. The organic solution wasdried (NaSO₄), filtered, concentrated, and crystallized (Et₂O/hexanes)to give a white solid, 110 mg (43%). ¹H-NMR (CDCl₃) δ 1.25 (s, 6H,2(CH₃)), 1.31 (s, 6H, 2(CH₃)), 1.70 (s, 4H, 2(CH₂)), 2.06 (s, 3H,Ar—CH₃), 2.14 (s, 3H, CH₃), 4.71 (s, 2H, OCH₂), 5.85 (s, 1H, ═CH), 7.01(s, 1H, Ar—CH), 7.16 (s, 1H, Ar—CH), 7.55 (d, 2H, Ar—CH₂, J=8.2 Hz),8.02 (d, 2H, Ar—CH₂, J=8.2 Hz).

EXAMPLE 55 Preparation of compound 143, where R₁, R₂, R₃, R₄, and R₅ aremethyl; R′ and R″ are 2-aminoethyloxime (Cl—NH₃+CH₂CH₂—O—N═); X=COOH(2-aminoethyloxime of 3-methyl-TTNCB)

To a solution of the oxime of 3-methyl-TTNCB (compound number 112, 108mg, 0.30 mmol) and 2-bromoethylamine hydrobromide (182 mg, 0.89 mmol) inDMF (1 mL) was added excess powered KOH at 0° C. The yellow solution wasallowed to warm to room temperature and stirred for 48 hr. Aqueous,saturated NH₄Cl (5.0 mL) was added and the aqueous layer was adjusted topH=2 with 1 M aqueous HCl. The aqueous solution was extracted 3 timeswith EtOAc; the organic layers were combined, and washed with water (2×)and brine. The organic solution was dried (MgSO₄), filtered, andconcentrated, and recrystallized (Et₂O/hexanes) to give a white solid,64 mg (50%). IR (KBr pellet) 3600-3200 broad, 3420, 2922, 2855, 1695,1591, 1456, 1364, 1231, 1017 cm⁻¹; ¹H-NMR (CDCl₃/CD₃OD) δ 1.21 (s, 6H,2(CH₃)), 1.29 (s, 6H, 2(CH₃)), 1.70 (s, 4H, 2 (CH₂)), 2.06 (s, 3H,Ar—CH₃), 2.14 (s, 3H, CH₃), 3.29 (m, 2H, CH₂), 4.37 (m, 2H, CH₂), 7.00(s, 1H, Ar—CH), 7.24 (s, 1H, Ar—CH), 7.50 (d, 2H, Ar—CH₂, J=8.0 Hz),7.94 (d, 2H, Ar—CH₂, J=8.0 Hz).

Evaluation of Retinoid Receptor Subtype Selectivity

Representative synthetic retinoid compounds of the current inventionwere analyzed and found to exhibit subtype selectivity for retinoidreceptors, and to be capable of modulating processes selectivelymediated by retinoid X receptors, as discussed more fully below.

As employed herein, the phrase “processes selectively mediated byretinoid X receptors” refers to biological, physiological,endocrinological, and other bodily processes which are mediated byreceptors or receptor combinations which are responsive to retinoid Xreceptor selective processes, e.g., compounds which selectively activateone and/or multiple members of the RXR subfamily. Modulation includesactivation or enhancement of such processes as well as inhibition orrepression, and can be accomplished in vitro or in vivo. In vivomodulation can be carried out in a wide range of subjects, such as, forexample, humans, rodents, sheep, pigs, cows, and the like. It is wellaccepted that modulation of such processes has direct relevance to usein treating disease states.

The receptors which are responsive to retinoid X receptor selectiveligands include: retinoid X receptor-alpha, retinoid X receptor-beta,retinoid X receptor-gamma, and splicing variants encoded by the genesfor such receptors, as well as various combinations thereof (i.e.,homodimers, homotrimers, heterodimers, heterotrimers, and the like).Also included are combinations of retinoid X receptors with othermembers of the steroid/thyroid superfamily of receptors with which theretinoid X receptors may interact by forming heterodimers,heterotrimers, and the higher heteromultimers. For example, the retinoicacid receptor-alpha, -beta, or -gamma isoforms form a heterodimer withany of the retinoid X receptor isoforms, (i.e., alpha, beta, or gamma,including any combination of the different receptor isoforms), and thevarious retinoid X receptors form a heterodimer with thyroid receptorand form a heterodimer with vitamin D receptor. Members of the retinoidX receptor subfamily form a heterodimer with certain “orphan receptors”including PPAR (Issemann and Green, Nature, 347:645-49 (1990)); HNF4(Sladek et al., Genes & Development 4:2353-65 (1990)); the COUP familyof receptors (e.g., Miyajima et al., Nucleic Acids Research 16:11057-74(1988), and Wang et al., Nature, 340:163-66 (1989)); COUP-like receptorsand COUP homologs, such as those described by Mlodzik et al. (Cell,60:211-24 (1990)) and Ladias et al. (Science, 251:561-65 (1991)); andthe ultraspiracle receptor (e.g., Oro et al., Nature, 347:298-301(1990)). As employed herein, the phrase “members of the steroid/thyroidsuperfamily of receptors” (also known as “nuclear receptors” or“intracellular receptors”) refers to hormone binding proteins thatoperate as ligand-dependent transcription factors. Furthermore, thisclassification includes identified members of the steroid/thyroidsuperfamily of receptors for which specific ligands have not yet beenidentified (referred to hereinafter as “orphan receptors”). All membersof the intracellular receptor superfamily have the intrinsic ability tobind to specific DNA sequences. Following binding, the transcriptionalactivity of a target gene (i.e., a gene associated with the specific DNAsequence) is modulated as a function of the ligand bound to thereceptor. Also, see Heyman et al., Cell, 68:397-406 (1992), andcopending U.S. Ser. No. 809,980, filed Dec. 18, 1991, whose entiredisclosures are incorporated herein by reference.

The modulation of gene expression by the ligand retinoic acid and itsreceptors can be examined in a reconstituted system in cell culture.Such a system was used to evaluate the synthetic retinoid compounds ofthis invention for their interaction with the retinoid receptor subtypesRARα, RARβ, RARγ, RXRα, RXRβ, and RXRγ.

The system for reconstituting ligand-dependent transcriptional control,which was developed by Evans et al., Science, 240:889-95 (1988), hasbeen termed a “co-transfection” or “cis-trans” assay. This assay isdescribed in further detail in U.S. Pat. Nos. 4,981,784 and 5,071,773,which are incorporated herein by reference. Also see Heyman et al.,Cell, 68:397-406 (1992). The co-transfection assay provides a mechanismto evaluate the ability of a compound to modulate the transcriptionresponse initiated by an intracellular receptor. The co-transfectionassay is a functional, rapid assay that monitors hormone or ligandactivity, is a good predictor of an in vivo system, and can be used toquantitate the pharmacological potency and utility of such ligands intreating disease states. Berger et al., J. Steroid Biochem. Molec.Biol., 41:733-38 (1992).

Briefly, the co-transfection assay involves the introduction of twoplasmids by transient transfection into a retinoid receptor-negativemammalian cell background. The first plasmid contains a retinoidreceptor cDNA and directs constitutive expression of the encodedreceptor. The second plasmid contains a cDNA that encodes for a readilyquantifiable protein, e.g., firefly luciferase or chloramphenicol acetyltransferase (CAT), under control of a promoter containing a retinoidacid response element, which confers retinoid dependence on thetranscription of the reporter. In this co-transfection assay, allretinoid receptors respond to all-trans-retinoic acid in a similarfashion. This assay can be used to accurately measure efficacy andpotency of retinoic acid and synthetic retinoids as ligands thatinteract with the individual retinoid receptor subtypes.

Accordingly, synthetic retinoid compounds of the current invention wereevaluated for their interaction with retinoid receptor subtypes usingthe co-transfection assay in which CV-1 cells were co-transfected withone of the retinoid receptor subtypes, a reporter construct, and aninternal control to allow normalization of the response for transfectionefficiency. The following example is illustrative.

EXAMPLE 56

Retinoids: All-trans-retinoic acid (RA) and 13-cis-retinoic acid(13-cis-RA) were obtained from Sigma. 9-cis-retinoic acid (9-cis-RA) wassynthesized as described in Heyman et al., Cell, 68:397-406 (1992).Retinoid purity was established as greater than 99% by reverse phasehigh-performance liquid chromatography. Retinoids were dissolved indimethylsulfoxide for use in the transcriptional activation assays.

Plasmids: The receptor expression vectors used in the co-transfectionassay have been described previously (pRShRAR-α: Giguere et al. (1987);pRShRAR-β and pRShRAR-γ: Ishikawa et al. (1990); pRShRXR-α: Mangelsdorfet al., (1990); pRSmRXR-βand pRSmRXR-γ: Mangelsdorf et al., Genes &Devel., 6:329-44 (1992)). A basal reporter plasmid Δ-MTV-LUC (Hollenbergand Evans, Cell, 55:899-906 (1988)) (1988)) containing two copies of theTRE-palindromic response element 5′-TCAGGTCATGACCTGA-3′ (Umesono et al.,Nature, 336:262-65 (1988)) was used in transfections for the RARs, andCRBPIIFKLUC, which contains an RXRE (retinoid X receptor responseelement (Mangelsdorf et al., Cell, 66:555-61 (1991)), was used intransfections for the RXRs.

Co-transfection Assay In CV-1 Cells: A monkey kidney cell line, CV-1,was used in the cis-trans assay. Cells were transfected with twoplasmids. The trans-vector allowed efficient production of the retinoidreceptor in these cells, which do not normally express this receptorprotein. The cis-vector contains an easily assayable gene product, inthis case the firefly luciferase, coupled to a retinoid-responsivepromoter, i.e., an RARE or RXRE. Addition of retinoic acid or anappropriate synthetic retinoid results in the formation of aretinoid-RAR or —RXR complex that activates the expression of luciferasegene, causing light to be emitted from cell extracts. The level ofluciferase activity is directly proportional to the effectiveness of theretinoid-receptor complex in activating gene expression. This sensitiveand reproducible co-transfection approach permits the identification ofretinoids that interact with the different receptor isoforms.

Cells were cultured in DMEM supplemented with 10% charcoalresin-stripped fetal bovine serum, and experiments were conducted in96-well plates. The plasmids were transiently transfected by the calciumphosphate method (Umesono and Evans, Cell, 57:1139-46 (1989) and Bergeret al., J. Steroid Biochem. Molec. Biol., 41:733-38 (1992)) by using 10ng of a pRS (Rous sarcoma virus promoter) receptor-expression plasmidvector, 50 ng of the reporter luciferase (LUC) plasmid, 50 ng ofpRSβ-GAL(β-galactosidase) as an internal control, and 90 ng of carrierplasmid, pGEM. Cells were transfected for 6 h and then washed to removethe precipitate. The cells were then incubated for 36 h with or withoutretinoid. After the transfection, all subsequent steps were performed ona Beckman Biomek Automated Workstation. Cell extracts were prepared,then assayed for luciferase and β-galactosidase activities, as describedby Berger et al. (1992). All determinations were performed in triplicatein two independent experiments and were normalized for transfectionefficiency by using β-galactosidase as the internal control. Retinoidactivity was normalized relative to that of all-trans-retinoic acid andis expressed as potency (EC₅₀), which is the concentration of retinoidrequired to produce 50% of the maximal observed response, and efficacy(%), which is the maximal response observed relative to that ofall-trans-retinoic acid at 10⁻⁵ M. The data obtained is the average ofat least four independent experiments. Efficacy values less than 5% arenot statistically different than the 0% background. Compounds with anefficacy of less than 20% at concentrations of 10⁻⁵ M are considered tobe inactive. At higher concentrations of compound, such as 10⁻⁴ M, thesecompounds are generally toxic to cells and thus the maximal efficacy at10⁻⁵ M is reported in the tables and figures contained herein.

The synthetic retinoid compound 3-methyl-TTNCB, as described above, wasevaluated for its ability to regulate gene expression mediated byretinoid receptors. As shown in FIG. 1, this compound is capable ofactivating members of the RXR subfamily, i.e., RXRα, RXRβ, and RXRγ, butclearly has no significant activity for members of the RAR subfamily,i.e., RARα, RARβ, and RARγ. Assays using all-trans-retinoic acid (FIG.2) and 9-cis-retinoic acid (FIG. 3) were run for reference, anddemonstrate that these retinoic acid isomers activate members of boththe RAR and RXR subfamilies.

Potency and efficacy were calculated for the 3-methyl-TTNCB compound, assummarized in the following table. For reference, the data for9-cis-retinoic acid are also included. TABLE 1 Potency (nM) Efficacy3-Methyl-TTNCB RXRα 330 130% RXRβ 200 52% RXRγ 260 82% RARα >10,000  <2%RARβ >10,000  <4% RARγ >10,000  <4% 9-cis-retinoic acid RXRα 150 140%RXRβ 100 140% RXRγ 110 140% RARα 160 100% RARβ 5 82% RARγ 47 120%

As shown by the data in Table 1,3-methyl-TTNCB readily and at lowconcentrations activates RXRs. Further, 3-methyl-TTNCB is more potent anactivator of RXRs than RARs, and preferentially activates RXRs incomparison to RARs, in that much higher concentrations of the compoundare required to activate the RARs. In contrast, 9-cis-retinoic acid doesnot preferentially activate the RXRs, as also shown in Table 1. Rather,9-cis-retinoic acid activates the RARβ and RARγ isoforms at lowerconcentrations and more readily than the RXRβ and RXRγ isoforms, and hassubstantially the same, within the accuracy of the measurement, activityfor the RARα isoform in comparison to the RXRα isoform.

An extract reported to contain 9-cis-retinoic acid has previously beenreported as at least 10-fold more potent in inducing RXRα than RARα(Heyman et al., Cell, 68:397,399 (Jan. 24, 1992)). Presently availabledata indicate that 9-cis-retinoic acid does not preferentially activateRXRs in comparison to RARs, as shown and discussed above. The compoundsof this invention preferentially activate RXRs in comparison to RARs,and are preferably at least three times more potent as activators ofRXRs than RARs, and more preferably at least five times more potent asactivators of RXRs than RARs.

Potency and efficacy have also been calculated for the 3-methyl-TTNEB,3-bromo-TTNEB, 3-methyl-TTNCHBP, 3-methyl-TTNEHBP, TPNEP, and TPNCPcompounds, as summarized below in Table 2. TABLE 2 Potency (nM) Efficacy3-Methyl-TTNEB RXRα 40 83% RXRβ 21 102% RXRγ 34 80% RARα >10,000 6%RARβ >10,000 17% RARγ >10,000 19% 3-Bromo-TTNEB RXRα 64 88% RXRβ 54 49%RXRγ 52 71% RARα >10,000 3% RARβ >10,000 18% RARγ >10,000 15%3-Methyl-TTNCHBP RXRα 1100 113% RXRβ 1100 155% RXRγ 300 128%RARα >10,000 <2% RARβ >10,000 7% RARγ >10,000 17% 3-Methyl-TTNEHBP (63)RXRα 140 125% RXRβ 71 121% RXRγ 48 163% RARα >10,000 <2% RARβ 1,900 25%RARγ >10,000 10% TPNEP (58) RXRα 5 75% RXRβ 5 138% RXRγ 6 100%RARα >10,000 <2% RARβ >10,000 <2% RARγ 1,500 24% TPNCP (62) RXRα 4 63%RXRβ 4 93% RXRγ 3 49% RARα >10,000 <2% RARβ >10,000 <2% RARγ >10,000 <2%

As shown by the data in Table 2, 3-methyl-TTNEB, 3-bromo-TTNEB,3-methyl-TTNCHBP, 3-methyl-TTNEHBP, TPNEP, and TPNCP each readily andpreferentially activate the RXRs, and are more potent as activators ofRXRs than of RARs. The diminished activity of these compounds for theRARs in comparison to the RXRs is also shown for some of these compoundsin FIGS. 4-7.

The potency and efficacy of the oxime derivative compounds 112 and 115have also been calculated, as summarized below in Table 3. TABLE 3Potency (nM) Efficacy Oxime Cd. 112 RXRα 15 66% RXRβ 8 51% RXRγ 12 62%RARα >10,000 3% RARβ >10,000 3% RARγ >10,000 3% Oxime Cd. 115 RXRα 5 61%RXRβ 5 71% RXRγ 5 70% RARα >10,000 4% RARβ >10,000 2% RARγ >10,000 3%

As shown, oxime compounds 112 and 115 preferentially activate RXRs incomparison to RARs.

The selective activity of the compounds of this invention for Retinoid XReceptors is not exhibited by other known compounds. For example,compounds such as those described in U.S. Pat. No. 4,833,240 (Maignan etal.) appear structurally similar to the compounds of this invention, butlack a functional group (such as methyl, ethyl, isopropyl, bromo,chloro, etc.) at the 3-position. Such compounds have little or nopotency and lack any selectivity for RXRs.

For example, a representative compound of U.S. Pat. No. 4,833,240(Maignan) is shown below, along with the compound 3-methyl-TTNCB of thisinvention.

The potency and efficacy of the Maignon compound and that of3-methyl-TTNCB are summarized below: Potency (nM) Efficacy Maignon Ex.II RXRα 3,000 82% RXRβ 3,000 44% RXRγ 3,000 64% RARα >10,000 11% RARβ1,900 58% RARγ 2,000 56% 3-Methyl-TTNCB RXRα 330 130% RXRβ 200 52% RXRγ260 82% RARα >10,000 <2% RARβ >10,000 <4% RARγ >10,000 <4%

As shown, the Maignon compound is virtually inactive and shows noselectivity for RXRS. In contrast, the compounds of this invention suchas 3-methyl-TTNCB, which have a substituent at the 3-position, arepotent activators of RXRs and exhibit the unexpected RXR selectivityshown in Table 1 (as well as Tables 2 and 3) and discussed above.

It can be expected that synthetic retinoid ligands, such as thoseexemplified in Tables 1, 2, and 3 which preferentially affect some butnot all of the retinoic acid receptor isoforms, can, in pharmacologicalpreparations, provide pharmaceuticals with higher therapeutic indicesand a better side effect profile than currently used retinoids. Forexample, the compounds of the present invention have been observed to beless irritating to the skin than previously known retinoids.

The retinoid compounds of this invention are useful for the treatment ofcertain dermatological conditions such as keratinization disorders,i.e., differentiation/proliferation. A standard assay to determine theactivity of these compounds is the measurement of the enzymatic activityfor transglutaminase; this is a measure of the antiproliferative actionof retinoids. Retinoids have been shown to inhibit the pathway ofdifferentiation, which is indicated by a decrease in several biochemicalmarkers that are associated with the expression of squamous cellpheno-type, such as transglutaminase. (Yuspa et al., Cancer Research,43:5707-12 (1983)). As can be seen from FIG. 8, the 3-methyl-TTNCBcompound is capable of inhibiting transglutaminase activity and inhibits50% of the enzyme activity at 1×10⁻⁷ M.

The retinoid compounds of this invention have been demonstrated in invitro tests to block (or antagonize) AP-1 activity, a set of oncogeneswhich drive cellular proliferation. Many proliferative disorders are theresults of oncogenes/oncogene activation, and therefore a compound whichblocks the AP-1 oncogene pathway can be used to treat diseasesassociated with proliferative disorders including cancers, inflammatorydiseases, psoriasis, etc. For example, the compound 3-methyl-TTNEB wasevaluated using the co-transfection assay in which HeLa cells wereco-transfected with a plasmid expressing RXRα under the control of aconstitutive promoter, and a plasmid which expresses the reporter enzymeluciferase under the control of a conditional promoter (collagenase)containing an AP-1 responsive element. E.g., Angel et al., Mol. Cell.Biol., 7:2256 (1987); Lafyatis et al., Mol. Endocrinol., 4:973 (1990).Following AP-1 activation, the assay results showed antagonism of AP-1activity by the 3-methyl-TTNEB compound via RXRα in a dose-dependentmanner. Other compounds of this invention have shown similar antagonismof AP-1 activity. These results demonstrate that RXR-selective compoundssuch as 3-methyl-TTNEB can be used as anti-proliferatives to limit cellgrowth and treat diseases associated with hyperproliferation.

The compounds of this invention also exhibit good comedolytic activityin the test on Rhino mice described by Kligman et al. (J. of Inves.Derm., 73:354-58 (1979)) and Mezick et al. (J. of Inves. Derm.,83:110-13 (1984)). The test on Rhino mice has been a model for screeningcomedolytic agents. The activity of the 3-methyl-TTNCB retinoidcompound, as well as 9-cis and all-trans retinoic acid is shown in FIG.9. A 0.1% solution of 3-methyl-TTNCB is capable of inhibiting theutriculi diameter by approximately 50%. It has also been observed that3-methyl-TTNCB is less irritating to the skin of Rhino mice than 9-cis-or all-trans-retinoic acid.

The co-transfection assay allows examination of the ability of acompound to modulate gene expression in a retinoid receptor dependentfashion. To examine the ability of the compounds of this invention todirectly interact with the receptors, we have examined the ligandbinding properties of all six retinoid receptors. The receptors wereexpressed employing a baculovirus expression system in which we havedemonstrated that RXRα binds 9-cis-retinoic acid with high affinity(Heyman et al., Cell, 68:397 (1992)). The binding parameters ofreceptors expressed in baculovirus systems and mammalian systems areessentially identical.

The synthetic retinoids of the current invention have also been testedusing radioligand displacement assays. By testing the abilities ofvarious synthetic retinoids to compete with the radiolabeled retinoicacid for binding to various receptor isoforms, the ability of thecompounds to directly interact with the receptor can be examined, andthe relative dissociation constant for the receptor itself can bedetermined. This is an important supplementary analysis to theco-transfection assay since it can detect differentproperties/determinants of retinoid activity than are measured in theco-transfection assay. These determinants/differences in the two assaysystems may include (1) activating or inactivating metabolic alterationsof the test compounds, (2) binding to serum proteins which could alterthe free concentration or other properties of the test compound, (3)differences in cell permeation among test compounds, (4) intrinsicdifferences in the affinity of the test compounds for the receptorproteins, i.e., in K_(d), can be directly measured, and (5)conformational changes produced in the receptor after binding of thetest compound, reflected in the effects on reporter gene expression;(i.e., a functional measurement of receptor activation).

The 3-methyl-TTNCB compound is capable of displacing ³H-9-cis-retinoicacid bound to the RXRs, but is not capable of displacing radiolabeledligand that is bound to the RARs. This indicates that the 3-methyl-TTNCBcompound preferentially binds RXRs in comparison to RARs, a propertywhich would be expected of a ligand selective for the RXRs.

The K_(d) values were determined by application of the Cleng-Prusoffequation. These values were based on a determination of the IC₅₀ valueas determined graphically from a log-logit plot of the data.

Binding data were obtained for various compounds using the methoddiscussed in Wecksler & Norman, Anal. Biochem. 92:314-23 (1979). Resultsare shown below in Table 4.

The compounds in Table 4 were shown to readily and preferentiallyactivate RXRs, and to be more potent as activators of RXRs than of RARsusing the co-transfection assay, as discussed above. The binding resultsin Table 4 show these compounds to also preferentially bind RXRs versusRARs. TABLE 4 Binding (Kd₅₀ nM) 3-Methyl-TTNCB RXRα 350 RXRβ 230 RXRγ365 RARα >10,000 RARβ >10,000 RARγ >10,000 3-Methyl-TTNEB RXRα 41 RXRβ20 RXRγ 22 RARα 5,500 RARβ 5,400 RARγ 3,200 TPNEP (58) RXRα 22 RXRβ 21RXRγ 39 RARα 7,800 RARβ 4,900 RARγ 6,000 TPNCP (62) RXRα 3 RXRβ 3 RXRγ 3RARα >10,000 RARβ >10,000 RARγ >10,000 Oxime Cd. 112 RXRα 6 RXRβ 5 RXRγ5 RARα 8,700 RARβ >10,000 RARγ >10,000Taken together the ligand binding properties of these compounds andtheir ability to selectively modulate members of the RXR subfamilydemonstrate the identification of a class of compounds with the uniquebiological properties. The binding properties and especially thetranscriptional activation assays are a good predictor of thepharmacological activity of a compound (Berger et al. (1992)).

It has been recognized that the co-transfection assay provides afunctional assessment of the ligand being tested as either an agonist orantagonist of the specific genetic process sought to be affected, and isa predictor of in vivo pharmacology (Berger et al. (1992)). Ligandswhich do not significantly react with other intracellular receptors, asdetermined by the co-transfection assay, can be expected to result infewer pharmacological side effects. Because the co-transfection assay isrun in living cells, the evaluation of a ligand provides an earlyindicator of the potential toxicity of the candidate at concentrationswhere a therapeutic benefit would be expected.

Processes capable of being modulated by retinoid receptors, inaccordance with the present invention, include in vitro cellulardifferentiation, the regulation of morphogenetic processes includinglimb morphogenesis, regulation of cellular retinol binding protein(CRBP), and the like. As readily recognized by those of skill in theart, the availability of ligands for the retinoid X receptor makes itpossible, for the first time, to elucidate the processes controlled bymembers of the retinoid X receptor subfamily. In addition, it allowsdevelopment of assays for the identification of antagonists for thesereceptors.

The processes capable of being modulated by retinoid receptors, inaccordance with the present invention, further include the in vivomodulation of lipid metabolism; in vivo modulation of skin relatedprocesses (e.g., acne, psoriasis, aging, wrinkling, and the like); invivo modulation of programmed cell death (apoptosis); in vivo modulationof malignant cell development, such as occurs, for example, in acutepromyelocytic leukemia, mammary cancer, prostate cancer, lung cancer,cancers of the aerodigestive pathway, skin cancer, bladder cancer, andsarcomas; in vivo modulation of premalignant lesions, such as occurswith oral leukoplakia and the like; in vivo modulation of auto-immunediseases such as rheumatoic arthritis; in vivo modulation of fatty acidmetabolism; and the like. Such applications can be expected to allow themodulation of various biological processes with reduced occurrence ofundesirable side effects such as teratogenic effects, skin irritation,mucosal dryness, lipid disturbances, and the like. In vivo applicationscan be employed with a wide range of subjects, such as, for example,humans, rodents, sheep, pigs, cows, and the like.

For example, regarding the in vivo modulation of lipid metabolismreferred to above, apolipoprotein A-1 (“apoA1”) is a major proteincomponent of plasma high density lipoprotein (HDL) cholesterol. Thecirculating level of HDL in humans has been shown to be inverselycorrelated to the risk of atherosclerotic cardiovascular disease(ASCVD), the leading cause of morbidity and mortality in the UnitedStates, with a 3-4% increase in ASCVD for every 1% decrease in HDLcholesterol. Gordon et al., New Engl. J. Med., 321:1311 (1989). Whilethere are currently no good therapeutic regimens that increase HDLcholesterol, it can be expected that regulating synthesis of apoA1 canbe utilized to affect plasma concentrations of HDL cholesterol and todecrease the risk of ASCVD. Rubin et al., Nature, 353:265 (1991).

It has been established that regulation of transcription of apoA1 iscontrolled by members of the intracellular receptor superfamily, andfurther that the apoA1 gene transcription start site “A” is a highlyselective retinoic acid-responsive element that responds to retinoid Xreceptors. Rottman et al., Mol. Cell. Biol., 11:3814-20 (1991). BecauseRXRs can form heterodimers with transrepressers such as ARP-1 andCOUP-TF and transactivators such as HNF-4, and an RXR response elementresides in the apoA1 promoter, retinoids or ligands which selectivelyactivate members of the RXR family of retinoic acid receptors mayregulate apoA1 transcription. We have demonstrated in in vivo studiesthat ligands of this invention which have selective activity for RXRscan be used to modulate apoA1/HDL cholesterol and to significantly raiseplasma HDL levels, as demonstrated in the following example.

EXAMPLE 57

Male Sprague-Dawley rats (160-200 gram) were obtained from Harlan.Animals were fed standard laboratory diets (Harlan/Teklad) and kept inan environmentally controlled animal house with a light period lastingfrom 6 a.m. to 6 p.m. Animals were treated with drugs prepared assuspensions in olive oil.

To verify that RXR activation can increase plasma apoA1/HDL cholesterol,an initial study was carried out that included dosing rats for 4 dayswith an RAR-selective compound, all-trans retinoic acid, thenon-selective RAR/RXR agonist, 9-cis-retinoic acid, and either of twoRXR-selective agents, 3-methyl-TTNCB or 3-methyl-TTNEB. Each drug wasadministered at a dose of 100 mg/kg, i.p. Positive control groupsreceived olive oil as a vehicle. Twenty-four hours after the lasttreatment, rats were sacrificed by CO₂ inhalation, blood was collectedfrom the inferior vena cava into a tube containing 0.1 ml of 0.15% EDTAand centrifuged at 1500×g for 20 min. at 4° C. Plasma was separated andstored at 4° C. for evaluation of plasma total cholesterol and highdensity lipoprotein cholesterol (HDL-cholesterol).

Plasma total cholesterol was measured enzymatically utilizing BoeringerMannheim Diagnostics High Performance Cholesterol Methods with an ABBOTTVP Bichromatic Analyzer. HDL cholesterol was measured after preparationof the HDL-containing fraction by heparin-manganese precipitation ofplasma. HDL-cholesterol in this fraction was estimated as mentionedearlier. All HDL separations were checked for contamination by otherlipoproteins with agarose gel electrophoresis.

The results of this study are shown in FIG. 10. As shown, rats receivingthe RXR-selective compounds exhibited substantial and statisticallysignificant increases in HDL levels, particularly when receiving3-methyl-TTNEB.

Because the RXR-selective ligand 3-methyl-TTNEB was the mostefficacious, additional 4 day experiments were conducted with this agentat doses of 0.3, 1, 3, 6, 10, 30, 100, or 300 mg/kg i.p. in 1.0 ml oliveoil or 1, 3, 10, 30, 100, 300 mg/kg p.o. in 1.0 ml olive oil for 4 days.An additional 30 day p.o. study was conducted with 10, 30, or 100 mg/kg3-methyl-TTNEB to determine whether tolerance would develop to itspharmacological actions. For the rats receiving 3-methyl-TTNEB invarious doses for four days, it was observed that 3-methyl-TTNEBincreased plasma concentrations of HDL-cholesterol in a dose-dependentmanner with significant increases being made at the lowest dose, 0.3mg/kg i.p. At its optimally efficacious dose, 3-methyl-TTNEB increasedplasma HDL-cholesterol concentrations from 58 mg/dl to 95 mg/dl—anincrease of greater than 60%. Measurement of total cholesterol showed anincrease due to the increase of the HDL-cholesterol fraction.Measurement of triglycerides showed either no change or a slightdecrease. The 30-day study with 3-methyl-TTNEB did not indicatedevelopment of tolerance to its pharmacological action.

This study demonstrated that 3-methyl-TTNEB increased plasmaconcentrations of HDL-cholesterol in a dose-dependent manner followingeither i.p. or p.o. administration. The effect on circulating apoA1 oforally administered 3-methyl-TTNEB was also studied. Male Sprague-Dawleyrats were treated daily with 3 mg/kg body weight of 3-methyl-TTNEB forfour days. Serum samples were taken and analyzed by Western Blot usingantisera specific to rat apoA1. The treatment with 3-methyl-TTNEBresulted in a significant increase in circulating apoA1 level.

These studies demonstrate that treatment with an RXR specific compoundsuch as 3-methyl-TTNEB increases plasma concentrations of apoA1/HDLcholesterol. Since such animal studies are an accepted predictor ofhuman response, it would therefore be expected that such compounds couldbe used to therapeutically increase HDL-cholesterol in patients whoeither have, or are at risk for, atherosclerosis.

Additional in vitro studies were also performed utilizing theco-transfection assay previously described within this application todemonstrate the effect of RXR-selective ligands on regulation oftranscription of apoA1, as described in the following example.

EXAMPLE 58

This work focused on studying the transcriptional properties of theretinoid receptors RAR and RXR on a reporter molecule (e.g., luciferase)under control of a basal promoter containing the RXR response elementfrom the apoA1 gene (“A” site). Plasmid constructs coding for thevarious receptors were transfected into a human hepatocyte cell line(HepG-2) along with the reporter plasmid. Reporter plasmids containedmultimers of the apoA1 “A” site (−214 to −192 relative to transcriptionstart site) shown to bind RXR. Widom et al., Mol. Cell. Biol. 12:3380-89(1992); Ladias & Karathanasis, Science 251:561-65 (1991). Aftertransfection, treatment, harvest, and assay, the data obtained wasnormalized to transfected beta-galactosidase activity so as to controlfor transfection efficiency. The results demonstrated activation in thesystem with the RXR-specific ligands 3-methyl-TTNCB and 3-methyl-TTNEBin a concentration-dependent fashion, demonstrating that the RXRspecific ligands could regulate the transcriptional properties via the“A” site from the apoA1 gene. These compounds had no effect when RAR wasused in the transfection, demonstrating receptor specificity. Thetranscriptional regulation by RXR was dependent on the presence of thehormone response element.

These in vivo and in vitro studies demonstrate that the RXR-selectivecompounds of this invention can be used to elevate apoA1/HDL cholesteroland in the therapeutic treatment of related cardiovascular disorders.

Regarding the modulation of programmed cell death (apoptosis), theretinoid compounds of this invention have been demonstrated to induceapoptosis in particular cell types including leukemic cells and squamousepithelial carcinomas. Normally in cells there is a fine balance betweencellular processes of proliferation, differentiation, and cell death,and compounds which affect this balance may be used to treat certaincancers. Specifically, the ability of 3-methyl-TTNEB to inducedifferentiation, inhibit proliferation, and induce apoptosis in an acutepromyelucytic leukemia cell line, HL60, was studied. Cellularproliferation was measured by a thymidine incorporation assay(Shrivastav et at., Gender Res., 40:4438 (1980)), and 3-methyl-TTNEB wasfound to have no effect on cellular proliferation. This contrastsall-trans-retinoic acid, which inhibits thymidine incorporation.Cellular differation was measured by the ability of the cells to reducenitroblue tetrazolium (NBT) (Breitman et al., Proc. Natl. Acad. Sci.,77:2936 (1980)), and 3-methyl-TTNEB was found not to induce unduedifferentiation. The EC₅₀ for 3-methyl-TTNEB mediated differentiationwas >1000 μM, compared to 2.0 μM for all-trans-retinoic acid. However,3-methyl-TTNEB was found to induce transglutaminase activity in the HL60cells (Murtaugh et al., J. Biol. Chem. 258:11074 (1983)) in aconcentration-dependent manner, which correlates with the induction ofapoptosis or programmed cell death. It was further found that3-methyl-TTNEB was able to induce apoptosis as measured by DNAfragmentation and morphological changes. Other retinoid compounds ofthis invention showed similar results, and similar results were alsoshown in other cell lines such as squamous epithelial cell lines andME180 cells, a human cervical carcinoma.

These results show that RXR specific compounds such as 3-methyl-TTNEBinduce apoptosis with a minimal direct effect on inhibition ofproliferation and differentiation induction. Compounds which are capableof inducing apoptosis have been shown to be effective in cancerchemotherapy (e.g., anti-hormonal therapy for breast and prostatecancer).

In contrast, in another cell type, retinoids have been shown to inhibitactivation driven T-cell apoptosis and 9-cis-retinoic acid wasapproximately ten fold more potent than all-trans-retinoic acids(Ashwell et al., Proceedings National Academy of Science, Vol. 90, p.6170-6174 (1993)). These data imply that RXRs are involved in thisevent. Thus retinoids could be used to block and/or immunomodulateT-cell apoptosis associated with certain disease states (e.g., AIDS).

It has been surprisingly found that the administration of a ligand whichhas specific activity for RXRs but essentially no activity for RARs, incombination with a ligand that has specific activity for RARs but notRXRs, provides a cellular response at extremely low dosages, dosages atwhich the ligands individually provide no significant response.Specifically, the concentration-related effect of an RXR-specific ligandand a RAR-specific ligand on proliferation of a myeloma cell line (RPMI8226) was studied in in vitro studies using a thymidine incorporationassay. This assay examines the incorporation of radiolabeled thymidineinto DNA, and by determining the ability of a compound to inhibitthymidine incorporation into DNA, provides a measure of cellproliferation. (L. M. Bradley, Selected Methods in Cellular Immunology,Ch. 10.1, pp. 235-38, Mishell & Shiigi (eds.), Freeman & Co., New York,1980). Compounds which inhibit cell proliferation have well-knownutility in the treatment of certain cancers.

As shown previously (Table 2), 3-methyl-TTNEB activates members of theRXR subfamily and has no significant activity for members of the RARsubfamily. Examination of the effects of 3-methyl-TTNEB on theproliferation of myeloma cells show a concentration dependent inhibitionof thymidine incorporation. The IC₅₀ (the concentration of3-methyl-TTNEB required to produce 50% inhibition of the maximalresponse) is 10⁻⁷ M, as shown in FIG. 11. Concentrations less than 10⁻⁸M provide essentially no effect on cell proliferation, as also shown inFIG. 11.

It is well known that the compound TTNEB activates members of the RARsubfamily and has no significant activity for members of the RXRsubfamily. The compound TTNEB is shown below, and its activity is shownin Table 5.

TABLE 5 TTNPB Potency (nM) Efficacy RXRα >10,000 <5% RXRβ >10,000 <5%RXRγ >10,000 <5% RARα 52 30 RARβ 4 40 RARγ 0.4 50

The effect of TTNPB on cell proliferation is shown in FIG. 11. The IC₅₀value of TTNPB is about 5×10 ⁻¹¹ M, and a concentration of less than10⁻¹¹ M produces essentially no effect on cell proliferation.

However, it has been found that when 3-methyl-TTNEB and TTNPB arepresent together, each at a concentration where the compound aloneproduces substantially no anti-proliferative effect, the combination ofthe two compounds effectively blocks cell proliferation. The combinationof the two compounds appears to produce a greater than additive, orsynergistic, effect.

For example, as shown in FIG. 12, the presence of TTNPB at aconcentration of 10⁻¹¹ M produces a 9% inhibition on thymidineincorporation. However, combining it with 3-methyl-TTNEB at aconcentration of 10⁻⁸ M (which results in no effect on cellproliferation) produces a greatly enhanced inhibitory effect of 49%.Likewise, it has also been found that the inhibitory effect of3-methyl-TTNEB is greatly increased by the presence of TTNPB at aconcentration which alone produces no effect.

Since it is well-known that toxic side effects of compounds such asTTNPB are concentration-dependent, the synergistic effect resulting fromcombining such RAR-specific compounds with RXR-specific compounds can beexpected to permit lower dosages that are efficacious and to thereforereduce toxic side effects. For example, in cancer chemotherapy, use oftwo such compounds, in combination, at relatively low doses can beexpected to produce the desired beneficial effect, while minimizingundesired side effects which result at higher doses of the compounds.

In vitro studies utilizing the co-transfection assay have also shownthis same synergistic effect. For example, utilizing the co-transfectionassay described previously and employing RAR-α and RXR-α and a reporterconsisting of the ApoA1 response element “A” site in the context ofTKLUC (Ladias & Karathanasis, Science 251:561-65 (1991), transfectionswere performed in HEPG2 cells. In this study, 100 ng of the designatedreceptor were used and RSVCAT was used as a carrier to keep the amountof RSV promoter constant. All compounds were added at a finalconcentration of 10⁻⁷ M. The RXR specific compound 3-methyl-TTNEB (Table2, above) and the RAR specific compound TTNPB (Table 5, above) wereutilized. As shown below in Table 6, the relative normalized responseobserved utilizing the co-transfection assay also demonstrated asynergistic effect when a combination of the two compounds was utilized,compared to the response achieved utilizing the compounds individually.TABLE 6 Reporter Activity Compound (Fold Induction) 3-methyl-TTNEB 5TTNPB 32 3-methyl-TTNEB + TTNPB 75

As will be discernable to those skilled in the art from the foregoingdiscussions, the biological response of an RAR selective compound at agiven concentration can be synergistically enhanced by combining thecompound with an RXR selective compound. Similarly, the biologicalresponse of an RXR selective compound can be enhanced by combining thecompound with an RAR selective compound. Thus, it becomes possible toachieve a desirable biological response, using a combination of RAR andRXR selective compounds, at lower concentrations than would be the caseusing the compounds alone. Among the advantages provided by suchcombinations of RAR and RXR selective compounds are desirabletherapeutic effects with fewer side effects. In addition, novel effectsthat are not obtainable with either agent alone may be achieved bycombinations of RAR and RXR selective compounds.

It has been further demonstrated that RXR-specific compounds alsosynergistically enhance the response of other hormonal systems.Specifically, peroxisome proliferator-activated receptor (PPAR) is amember of the intracellular receptor super family that plays a role inthe modulation of lipid homeostasis. PPAR has been shown to beachemotivated by amphipathic carboxylates, such as clofibric acid, andgemfibrizol. These agents, called peroxisome proliferators, have beenused in man as hypolipidemic agents. The addition of 9-cis-retinoic acid(a retinoid ligand which activates both RAR and RXR receptors) andclofibric acid to HepG2 cells transfected with RXRα and PPAR expressionplasmids, results in the activation of receptor gene which was greaterthan the sum of the activation with each ligand separately. (Kliewer etal., Nature 358:771 (1992)). Similarly, when the above two receptorswere co-transfected into HepG2 cells, the addition of both anRXR-specific ligand (3-methyl-TTNEB) and clofibric acid was found toproduce a greater than additive response as determined by activation ofa target reporter gene, as shown below in Table 7. TABLE 7 CompoundNormalized Response (%) clofibric Acid 100 3-methyl-TTNEB 90 clofibricacid + 3- 425 methyl-TTNEB

A similar synergistic effect was observed with RXR and RXR-specificligands and the Vitamin D receptor (VDR) and its cognate ligands. WhenRXPβ and VD receptors were co-transfected into CV-1 cells containing ahormone response element, the addition of RXR selective 3-methyl-TTNCBand 1,25-dihydroxy-vitamin D (1,25-D) produced a greater than additiveresponse than was observed for each of the individual ligands, as shownbelow in Table 8. TABLE 8 Compound Normalized Response (%) 1,25-D 1003-Methyl-TTNCB 13 1,25-D + 3-methyl-TTNCB 190

As shown, the above results indicate that each pair of receptors(RXRα/PPAR and RXRβ/VDR, respectively), in the presence of ligands knownto specifically activate their respective receptors, are capable ofproducing a synergistic response. The results indicate that the responseof a single agent can be enhanced by the combination of the two agents,or that comparable biological or therapeutic responses can be achievedby use of lower doses of such agents in combination.

The observation that RXR-specific ligands are able to actsynergistically with RAR ligands, PPAR ligands, and Vitamin D ligandsindicates that RXR-specific ligands have usefulness not only as singletherapeutic agents but also in combination therapy to obtain enhancedbiological or therapeutic response by the addition of the RXR-specificligand. Such combination therapy also may provide an added benefit ofdecreasing the side effects associated with the primary agent byemploying lower doses of that agent. For example, use of Vitamin D or arelated Vitamin D receptor ligand in conjunction with an RXR selectivecompound for the treatment of a variety of disorders including skindiseases (acne, psoriasis), hyperproliferative disorders (benign andmalignant cancers) and disorders of calcium homeostasis may decrease theadverse side effects associated with Vitamin D therapy alone.

As a further example, the RXR-specific compounds of this invention havebeen demonstrated in vitro to act synergistically with compounds whichaffect cellular proliferation, such as Interferon. Specifically, thegrowth properties of two human tumor cell lines (ME180, a squamous cellcarcinoma, and RPMI18226, a multiple myeloma) were monitored in thepresence of the compound 3-methyl-TTNEB alone and in combination withInterferonα2b, utilizing standard cell culture procedures. The effectson growth of these cells were monitored by evaluation of cell number,and also by evaluation of growth in semi-solid medium for the RPMI18226cell line. Both 3-methyl-TTNEB and Interferonα2b were found to inhibitcell growth in a concentration-dependent manner, and each alone toproduce a significant depression in cell proliferation. In addition,when the cells were treated with both compounds, an additive or agreater than additive effect on the depression in cell proliferation wasobserved. Treatment with other chemotherapeutic agents includinganti-proliferative agents and/or cell-cycle modulators (e.g.,methotrexate, fluorouracil (5-FU), ARA-C, etc.) in combination withRXR-specific compounds would be expected to produce similar results. Theenhanced anti-proliferative effect can be expected to permit lowertherapeutic doses in treatment of proliferative disorders, such assquamous cell and other carcinomas. In addition, combination therapycould allow the use of lower doses of these compounds to achieve acomparable beneficial effect along with fewer side effects/toxiceffects, thereby enhancing the therapeutic index of the therapy. Thetherapeutic index is defined as the ratio of efficacy to toxicity of acompound.

Since RXR is known to form heterodimers with various members of theintracellular receptor super family, it can be expected that thesynergistic response observed with use of RXR-selective ligands may beachieved with other receptors with which heterodimers are formed. Theseinclude PPARs, RARs, Vitamin D, thyroid hormone receptors, HNF4, theCOUP family of receptors, as referenced above, and other as yetunidentified members of the intracellular super family of receptors.

As will be further discernible to those skilled in the art, thecompounds disclosed above can be readily utilized in pharmacologicalapplications where selective retinoid receptor activity is desired, andwhere it is desired to minimize cross reactivities with other relatedintracellular receptors. In vivo applications of the invention includeadministration of the disclosed compounds to mammalian subjects, and inparticular to humans.

The compounds of the present invention are small molecules which arerelatively fat soluble or lipophilic and enter the cell by passivediffusion across the plasma membrane. Consequently, these ligands arewell suited for administration orally and by injection, as well astopically. Upon administration, these ligands can selectively activateretinoid X receptors, and thereby selectively modulate processesmediated by these receptors.

The pharmaceutical compositions of this invention are prepared inconventional dosage unit forms by incorporating an active compound ofthe invention, or a mixture of such compounds, with a nontoxicpharmaceutical carrier according to accepted procedures in a nontoxicamount sufficient to produce the desired pharmacodynamic activity in amammalian and in particular a human subject. Preferably, the compositioncontains the active ingredient in an active, but nontoxic, amountselected from about 5 mg to about 500 mg of active ingredient per dosageunit. This quantity depends on the specific biological activity desiredand the condition of the patient.

The pharmaceutical carrier or vehicle employed may be, for example, asolid or liquid. A variety of pharmaceutical forms can be employed.Thus, when using a solid carrier, the preparation can be plain milled,micronized in oil, tableted, placed in a hard gelatin or enteric-coatedcapsule in micronized powder or pellet form, or in the form of a troche,lozenge, or suppository. When using a liquid carrier, the preparationcan be in the form of a liquid, such as an ampule, or as an aqueous ornonaqueous liquid suspension. For topical administration, the activeingredient may be formulated using bland, moisturizing bases, such asointments or creams. Examples of suitable ointment bases are petrolatum,petrolatum plus volatile silicones, lanolin, and water in oil emulsionssuch as Eucerin (Beiersdorf). Examples of suitable cream bases are NiveaCream (Beiersdorf), cold cream (USP), Purpose Cream (Johnson & Johnson)hydrophilic ointment (USP), and Lubriderm (Warner-Lambert).

The following examples provide illustrative pharmacological compositionformulations:

EXAMPLE 59

Hard gelatin capsules are prepared using the following ingredients:Quantity (mg/capsule) 3-methyl-TTNCB 140 Starch, dried 100 Magnesiumstearate  10 Total 250 mgThe above ingredients are mixed and filled into hard gelatin capsules in250 mg quantities.

EXAMPLE 60

Quantity (mg/tablet) 3-methyl-TTNCB 140 Cellulose, microcrystalline 200Silicon dioxide, fumed 10 Stearic acid 10 Total 360 mg

The components are blended and compressed to form tablets each weighing360 mg.

EXAMPLE 61

Tablets, each containing 60 mg of active ingredient, are made asfollows: Quantity (mg/tablet) 3-methyl-TTNCB 60 Starch 45 Cellulose,microcrystalline 35 Polyvinylpyrrolidone (PVP) 4 (as 10% solution inwater) Sodium carboxymethyl starch (SCMS) 4.5 Magnesium stearate 0.5Talc 1.0 Total 150 mg

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution of PVP is mixed withthe resultant powders, which are then passed through a No. 14 mesh U.S.sieve. The granules so produced are dried at 50° C. and passed through aNo. 18 mesh U.S. sieve. The SCMS, magnesium stearate, and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

EXAMPLE 62

Suppositories, each containing 225 mg of active ingredient, may be madeas follows: 3-methyl-TTNCB   225 mg Saturated fatty acid glycerides2,000 mg Total 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of normal 2 g capacity and allowed to cool.

EXAMPLE 63

An intravenous formulation may be prepared as follows: 3-methyl-TTNCB100 mg Isotonic saline 1,000 ml Glycerol 100 ml

The compound is dissolved in the glycerol and then the solution isslowly diluted with isotonic saline. The solution of the aboveingredients is then administered intravenously at a rate of 1 ml perminute to a patient.

The compounds of this invention also have utility when labeled asligands for use in assays to determine the presence of RXRs. They areparticularly useful due to their ability to selectively bond to membersof the RXR subfamily and can therefore be used to determine the presenceof RXR isoforms in the presence of other related receptors.

Due to the selective specificity of the compounds of this invention forretinoid X receptors, these compounds can also be used to purify samplesof retinoid X receptors in vitro. Such purification can be carried outby mixing samples containing retinoid X receptors with one of more ofthe bicyclic derivative compounds disclosed so that the compound(ligand) binds to the receptor, and then separating out the boundligand/receptor combination by separation techniques which are known tothose of skill in the art. These techniques include column separation,filtration, centrifugation, tagging and physical separation, andantibody completing, among others.

While the preferred embodiments have been described and illustrated,various substitutions and modifications may be made thereto withoutdeparting from the scope of the invention. Accordingly, it is to beunderstood that the present invention has been described by way ofillustration and not limitation.

1-3. (canceled)
 4. A compound having the formula:

wherein: R₁ and R₂, each independently, represent hydrogen or loweralkyl or acyl having 1-4 carbon atoms; Y represents C, O, S, N, CHOH,CO, SO, SO₂; R₃ represents hydrogen or lower alkyl having 1-4 carbonatoms where Y is C or N; R₄ represents hydrogen or lower alkyl having1-4 carbon atoms where Y is C, but R₄ does not exist if Y is N, andneither R₃ or R₄ exist if Y is S, O, CHOH, CO, SO, or SO₂; R′ and R″represent hydrogen, lower alkyl or acyl having 1-4 carbon atoms, OH,alkoxy having 1-4 carbon atoms, thiol or thio ether, or amino, or R′ orR″ taken together form an oxo (keto), methano, thioketo, HO—N═, NC—N═,(R₇R₈)N—N═, R₁₇O—N═, R₁₇N═, epoxy, cyclopropyl, or cycloalkyl group andwherein the epoxy, cyclopropyl, and cycloalkyl groups can be substitutedwith lower alkyl having 1-4 carbons or halogen; R₅ represents hydrogen,a lower alkyl having 1-4 carbons, halogen, nitro, OR₇, SR₇, NR₇ R₈, or(CF)_(n)CF₃, but R₅ cannot be hydrogen if Z, Z′, Z″, and Z′″, are allcarbon, and R′ and R″ represent H, OH, C₁-C₄ alkoxy or C₁-C₄ acyloxy orR′ and R″ taken together form an oxo, methano, or hydroxyimino group; R₇represents hydrogen or a lower alkyl having 1-6 carbons; R₈ representshydrogen or a lower alkyl having 1-6 carbons; R₉ represents a loweralkyl having 1-4 carbons, phenyl, aromatic alkyl, or q-hydroxy-phenyl,q-bromophenyl, q-chlorophenyl, q-fluorophenyl, or q-iodophenyl, whereq=2-4; R₁₇ represents hydrogen, lower alkyl having 1-8 carbons, alkenyl(including halogen, acyl, OR₇ and SR₇ substituted alkenes), R₉, alkylcarboxylic acid (including halogen, acyl, OR₇ and SR₇ substitutedalkyls), alkenyl carboxylic acid (including halogen, acyl, OR₇ and SR₇substituted alkenes), alkyl amines (including halogen, acyl, OR₇ and SR₇substituted alkyls), and alkenyl amines (including halogen, acryl, OR₇and SR₇ substituted alkenes); X is COOH, tetrazole, PO₃H, SO₃H, CHO,CH₂OH, CONH₂, COSH, COOR₉, COSR₉, CONHR₉, or COOW where W is apharmaceutically acceptable salt, and where X can originate from any Cor N on the ring; Z, Z′, Z″, and Z′″, each independently, represent C,S, O, N, but one of Z, Z′, Z″, and Z′″ is not O or S if attached by adouble bond to another such Z or if attached to another such Z which isO or S, and is not N if attached by a single bond to another such Zwhich is N; n=0-3; and the dashed lines in the second and seventhstructures shown depict optional double bonds; or a pharmaceuticallyacceptable salt thereof. 5-13. (canceled)
 14. A pharmaceuticalcomposition, comprising: a pharmaceutically acceptable vehicle; and oneor more compounds of claim
 4. 15-18. (canceled)
 19. A method formodulating a process mediated by one or more Retinoid X Receptors, saidmethod comprising causing said process to be conducted in the presenceof at least one compound as set forth in claim
 4. 20. The method ofclaim 19, wherein said Retinoid X Receptor is Retinoid X Receptor-alpha,Retinoid X Receptor-beta, or Retinoid X Receptor-gamma.
 21. The methodof claim 19, wherein said process is the in vivo modulation of lipidmetabolism, in vivo modulation of skin-related processes, in vivomodulation of autoimmune diseases, in vivo modulation of fatty acidmetabolism, in vivo modulation of malignant cell development, in vivomodulation of premalignant lesions, or in vivo modulation of programmedcell death.
 22. The method of claim 21, wherein said process is the invivo enhancement of programmed cell death.
 23. The method of claim 21,wherein said process is the in vivo inhibition of programmed cell death.24. The method of claim 19, wherein said process is in vivo or in vitrocellular growth and differentiation, or in vivo limb morphogenesis.25-28. (canceled)
 29. A method for treating a mammalian subjectrequiring Retinoid X Receptor therapy, comprising administering to suchsubject a pharmaceutically effective amount of one or more compounds ofclaim
 4. 30. A method for increasing plasma concentrations of highdensity lipoprotein in a mammalian subjects comprising administering tosuch subject a pharmaceutically effective amount of one or morecompounds of claim
 4. 31-44. (canceled)
 45. The pharmaceuticalcomposition of claim 14, wherein the pharmaceutically acceptable vehicleis suitable for enteral, parenteral, or topical administration.
 46. Acompound selected from among2-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl)thiophene-5-carboxylicacid (TTNCTC) and2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl)thiophene-5-carboxylicacid (TTNETC).